Edible plant-derived microvesicle compositions for diagnosis and treatment of disease

ABSTRACT

Compositions that include a therapeutic agent encapsulated by an edible plant-derived microvesicle are provided. Methods for treating an inflammatory disorder and methods for treating a cancer are further provided and include administering an effective amount of a composition that includes a therapeutic agent encapsulated by an edible plant-derived microvesicle to a subject. Further provided are methods of diagnosing a colon cancer that include the steps of administering an edible plant-derived microvesicle incorporating a detectable label to a subject and then determining an amount of the detectable label in an intestine of the subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Pat. Application Serial No.16/383,085 (pending), filed Apr. 12, 2019, which itself is acontinuation of U.S. Pat. Application Serial No. 14/356,853 (abandoned),filed May 7, 2014, which itself is a U.S. National Stage application ofPCT International Patent Application Serial No. PCT/US2012/056298(expired), filed Sep. 20, 2012, which itself claims priority from U.S.Provisional Application Serial No. 61/556,565 (expired), filed Nov. 7,2011. The entire disclosure of each of these applications isincorporated herein by this reference.

TECHNICAL FIELD

The presently-disclosed subject matter relates to edible plant-derivedmicrovesicle compositions and methods of using the same for thediagnosis and treatment of disease. In particular, thepresently-disclosed subject matter relates to compositions that includetherapeutic agents encapsulated by edible plant-derived microvesiclesand that are useful in the diagnosis and treatment of disease.

BACKGROUND

Microvesicles are small assemblies of lipid molecules (50-1000 nm insize), which include, but are not limited to, exosomes, epididimosomes,argosomes, exosome-like vesicles, microparticles, promininosomes,prostasomes, dexosomes, texosomes, dex, tex, archeosomes, and oncosomes.Microvesicles can be formed by a variety of processes, including therelease of apoptotic bodies, the budding of microvesicles directly fromthe cytoplasmic membrane of a cell, and exocytosis from multivesicularbodies. For example, exosomes are commonly formed by their secretionfrom the endosomal membrane compartments of cells as a consequence offusion of multivesicular bodies with the plasma membrane. The MVBs areformed by inward budding from the endosomal membrane and subsequentpinching off of small vesicles into the luminal space. The internalvesicles present in the MVBs are then released into the extracellularfluid as so-called exosomes.

In addition to being formed by a variety of processes, microvesicles areproduced by a variety of eukaryotic cells, including plant cells, andthe release and uptake of these secreted membrane vesicles has beenshown to allow for the transfer of small packages of information(bioactive molecules) to numerous target cells. Indeed, the contents ofthese packages are enriched in proteins, lipids, and microRNAs, andrecent biological and proteomic studies of microvesicles have furtherrevealed the biological functions of microvesicles. From these studies,it appears that one of the major roles of microvesicles is the exchangeof information through their secretion, with the functional consequencesof such membrane transfers including the induction, amplification and/ormodulation of recipient cell function. In this regard, a number ofstudies have led to the idea that microvesicles are a common mode ofintercellular communication.

Despite the number of studies linking microvesicles to intracellularcommunication, however, to date, the use of microvesicles as anefficient and effective delivery vehicle has yet to be fully realizeddue, at least in part, to the inability to produce the large quantitiesof microvesicles that are needed for therapeutic applications and to theinability to effectively and efficiently utilize the microvesicles todeliver a therapeutic agent to target cells and tissues, while alsoretaining the biological activity of the therapeutic agents.

SUMMARY

The presently-disclosed subject matter meets some or all of theabove-identified needs, as will become evident to those of ordinaryskill in the art after a study of information provided in this document.

This Summary describes several embodiments of the presently-disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently-disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

The presently-disclosed subject matter includes microvesiclecompositions and methods of using the microvesicle compositions for thediagnosis and treatment of disease. More specifically, thepresently-disclosed subject matter relates to microvesicle compositionsthat include one or more therapeutic agents encapsulated by an edibleplant-derived microvesicle and that are useful in the diagnosis andtreatment of disease.

In some embodiments of the presently-disclosed subject matter, acomposition is provided that includes a therapeutic agent encapsulatedby an edible-plant derived microvesicle. In some embodiments, the edibleplant is a fruit, such as, in some embodiments, a grape, a grapefruit,or a tomato.

In some embodiments, a microvesicle composition is provided where thetherapeutic agent is selected from a phytochemical agent, a stat3inhibitor, and a chemotherapeutic agent. In some embodiments, thetherapeutic agent is a phytochemical agent, such as, in someembodiments, curcumin. In some embodiments, the therapeutic agent is aphytochemical agent selected from curcumin, resveratrol, baicalein,equol, fisetin, and quercetin. In other embodiments, the therapeuticagent is a Stat3 inhibitor, such as JSI-124. In further embodiments, thetherapeutic agent is a chemotherapeutic agent that, in certainembodiments, is selected from the group consisting of retinoic acid,5-fluorouracil, vincristine, actinomycin D, adriamycin, cisplatin,docetaxel, doxorubicin, and taxol.

In yet further embodiments of the compositions of thepresently-disclosed subject matter, the therapeutic agent comprises anucleic acid molecule selected from a siRNA, a microRNA, and a mammalianexpression vector. In some embodiments, the microvesicle composition isin the form of a pharmaceutical composition where the edible-plantderived microvesicle encapsulating the therapeutic agent is furthercombined with a pharmaceutically-acceptable vehicle, carrier, orexcipient.

Further provided, in some embodiments of the presently-disclosed subjectmatter, are methods for treating an inflammatory disorder. In someembodiments, a method for treating an inflammatory disorder is providedthat comprises administering to a subject in need thereof an effectiveamount of a composition that includes a therapeutic agent encapsulatedby an edible plant-derived microvesicle. In some embodiments, thecomposition is administered orally or intransally. In some embodiments,administering the edible-plant derived microvesicle composition reducesan amount of an inflammatory cytokine in a subject, including, in someembodiments, a reduction in an amount of tumor necrosis factor-α,interleukin-1β, interferon-γ, or interleukin-6.

In some embodiments of the presently-disclosed methods of treating aninflammatory disorder, the inflammatory disorder is selected fromsepsis, septic shock, colitis, colon cancer, and arthritis. In someembodiments, the inflammatory disorder is colitis, where, in certainembodiments, administering the composition increases an amount ofintestinal epithelial cell proliferation in a subject or increases anamount of Wnt-β-catenin signaling in the intestine of a subject tothereby treat the colitis.

Still further provided, in some embodiments of the presently-disclosedsubject matter, are methods for treating a cancer. In some embodiments,a method for treating a cancer in a subject is provided that comprisesadministering to a subject an effective amount of a compositionincluding a therapeutic agent encapsulated by a microvesicle, where themicrovesicle is derived from an edible plant. In some embodiments, thetherapeutic agent encapsulated by the edible-plant derived microvesicleis selected from a phytochemical agent, a stat3 inhibitor, and achemotherapeutic agent. In some embodiments, the microvesicles of thepresent compositions comprise a cancer targeting moiety for directingthe composition to a cancer cell. In some embodiments, the cancertargeting moiety comprises folic acid.

In some embodiments of the methods for treating a cancer disclosedherein, the methods are used to treat a brain cancer, a breast cancer, alung cancer, or a colon cancer. In some embodiments, the cancer is abrain cancer that, in some embodiments, comprises a glioma. In someembodiments, the microvesicle compositions of the presently-disclosedsubject matter are used to treat the cancer by administering themicrovesicle compositions intranasally, orally, or intratumorally.

Still further provided, in some embodiments of the presently-disclosedsubject matter, are methods for diagnosing a colon cancer. In someembodiments, a method for diagnosing a colon cancer in a subject isprovided that includes the steps of: administering an effective amountof a composition that includes an edible plant-derived microvesicleincorporating a detectable label; determining an amount of thedetectable label in an intestine of the subject; and comparing theamount of the detectable label, if present, to a control level of thedetectable label, such that the subject is then diagnosed as havingcolon cancer or a risk thereof if there is a measurable difference inthe amount of the detectable label as compared to the control level. Insome embodiments, the subject has colon cancer. In some embodiments,based on the determined amounts of detectable label, a treatment isfurther selected or modified. In some embodiments, the detectable labelcomprises a radioisotope or a fluorescent probe.

Further advantages of the presently-disclosed subject matter will becomeevident to those of ordinary skill in the art after a study of thedescription, Figures, and non-limiting Examples in this document.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are photographs showing the results of sucrose gradientdifferential centrifugation procedures used to isolate exosomes fromgrapes and grapefruits, including: a photograph of banded exosomes aftersucrose gradient centrifugation (FIG. 1A, middle band); electronmicroscopy photographs confirming the presence of grape-derivedexosome-like particles (GELPs, FIG. 1B) and grapefruit-derivedexosome-like particles (GFELPs, FIG. 1C); and a photograph showing theresults of an SDS-PAGE analysis of the GELPs and GFELPs, where 100 mg ofGELP, GFELP or TS/A exospore proteins were run on a 10% SDS PAGE (FIG.1D);

FIGS. 2A-2E include images and graphs showing the uptake of GELPs byvarious cells and tissues subsequent to oral administration of theGELPs, including: photographs showing the binding of GELPs in miceintestines after the mice were orally administered GELPs labeled usingan IRDye 800CW labeling kit, PBS diluted free IRDye 800CW dye used at anequal amount for labeling GELPs, or PBS, where the GELPS or free dyewere administered at a dose of 100 or 500 mg in 100 µl of PBS (FIG. 2A);graphs showing the fluorescence activated cell sorting (FACS) ofintestinal epithelial cells or peripheral blood for EPCAM⁺PHK67⁺ orCD14⁺PHK67⁺ cells within the gated R1 region, where mice were treatedwith GELP (200 mg), and where PHK67 dye was administered orally at dosesfrom 10 to 200 mg in 100 µl of PBS and the cells were collected threehours after administration (FIG. 2B); a graph showing the percent ofPKH67+ cells in the peripheral blood of mice as a function of the amountof GELP orally administered to each mouse (FIG. 2C); photographs showingsynovial tissues of mice orally administered 200 µg of GELPs or free dyeorally (FIG. 2D); and a graph and images showing the FACS analysis ofF4/80⁺PHK67⁺ cells (inset, right panel) in the gated R1 region (inset,left panel) of cells from synovial tissues of mice orally administeredGELP;

FIGS. 3A-3F includes images and graphs showing the attenuation ofcollagen II-induced arthritis in DBA/1j mice orally administered GELPsencapsulating curcumin (GELP- Cur), including: images of GELPS beforeand five minutes after co-incubation of 40 µl of 20 mM curcumin with 500µg GELP in 200 µl PBS at 22° C. (FIG. 3A), where afterultracentrifugation for 1.5 h, the GELP-Cur complex observed in thesucrose gradient (top of panel) was examined by electromicroscopy(bottom of panel); graphs showing the concentration of TNF-α (FIG. 3B)and IL-6 (FIG. 3C) in sera of mice twenty days after DBA/1j mice wereimmunized with collagen II, where mice were orally administered, everyother day for 20 days, GELP-Cur complex (4.0 mg/kg of curcuminencapsulated ), GELP at an equal amount used in GELP-Cur, free curcumin(4.0 mg/kg ), or PBS, and where blood was taken immediately before or 1h after treatments on days 1, 10, and 20; and graphs showing the levelsof TNF-α (FIG. 3D) and IL-6 (FIG. 3E) in supernatants of cell culturesof anticoagulated whole blood from the same mice as in FIGS. 3B and 3C;and a graph showing the correlation between the induction of IL-6 in thesera and the amount of CD14⁺PI⁺AnnexinV⁺ cells in the peripheral bloodof mice collected at day 1 after the foregoing treatments and analyzedusing a Spearman’s rank test (FIG. 3F);

FIG. 4 is a schematic diagram showing the protocols used forinvestigating colon carcinogenesis, where 7-week old female C57BL/6jmice were given a single dose of AOM (10 mg/kg/body wt) or PBS as acontrol by oral gavage and on the same day, mice were provided 2% DSS intheir drinking water for 7 days, followed by 2 weeks of untreated water,where the cycle was repeated two more times for a total of three cycles,and where, at the beginning of week 10, mice were treated withGFELP-Cur, GELP-Cur, or Cur every other day for three weeks;

FIGS. 5A-5C includes images and graphs showing the reduction of AOMinduced colon cancer in mice treated as described in FIG. 4 , including:an image of representative excised colons from mice treated withGFELP-cur, GELP-cur and free Cur (FIG. 5A); and an enzyme-linkedimmunosorbent assay (ELISA) analysis of the levels of MIP1 (FIG. 5B) andIL-6 (FIG. 5C) produced from the colon tissue of treated mice;

FIGS. 6A-6H include images and graphs showing the ability of GELPs toprotect mice against DSS induced colitis, including: an image showingC57BL/6 mice who were either provided 3.0% DSS in the drinking waterover the course of 7 days and developed severe colitis symptoms withintense depression (DSS+PBS) or were fed GELPs (DSS+GELPs) and exhibitedno visible symptoms (FIG. 6A); a graph showing the incidence of occultblood in fecal samples of DSS+PBS mice or DSS+GELPs mice as a functionof the number of days after DSS treatment (FIG. 6B); graphs showing thesurvival (FIG. 6C) and representative colon length (FIG. 6D) in the miceadministered DSS+PBS or DSS+GELPs; images and a graph showing thequantification of the villus length from the base of the villus to thevillus apex of GELP and PBS gavaged mice administered DSS (FIG. 6E); agraph showing the flux of 4-KDa FITC-dextran across colonic tissues inthe mice administered DSS (FIG. 6F); a graph showing the sera levels ofIL-6 and IL-1□ (FIG. 6G) in the mice administered DSS; and a graphshowing the amount of bacteria in the liver of the mice administered DSS(FIG. 6H);

FIGS. 7A-7G include images showing the increased expression andheterogeneous distribution of nuclear β-catenin in the intestine of micegavaged with PBS or GELPs; including images showing representativestaining for β-catenin from PBS or GELP gavaged mice at a lowmagnification (FIG. 7A, 10x) and a high magnification (FIG. 7B, rightcolumn), where the arrows indicate that the β-catenin positive cells; animage of a western blot of protein lysates from the intestines of PBS orGELP gavaged mice that were analyzed for ser 9 phosphorylated GSK3β,total GSK3β and β -actin (loading control) expression (FIG. 7C); andimages showing immunohistochemistry and western blot analysis of CT26(FIGS. 7D and 7F, respectively) or MCA38 (FIGS. 7E and 7G, respectively)in colon cells treated with GELPs (10 µg/ml) or BSA as control, where β-catenin expression and localization was determined viaimmunohistochemistry with β -catenin and is shown at low magnification(left panels) or increased magnification (right panels), and where totalprotein from lysates of each treatment was analyzed for ser 9phosphorylated GSK3β, total GSK3β, and β-actin (loading control)expression;

FIGS. 8A-8C are images showing the induction of BMI1 positive stem cellsin the intestine of mice gavaged with GELPs, including: images showingBMI1 positive stained cells (FIG. 8A) or BRDU + proliferative cells(FIG. 8B); and an image showing a representative haematoxylin and eosinstaining of crypts from PBS or GELP gavaged mice (FIG. 8C);

FIGS. 9A-9B include an image and a graph showing the use of DIRdye-labeled GELPs as an early indicator for AOM induced intestineleisure, including: representative photographs from PBS or AOM injectedmice (FIG. 9A); and a graph showing the signal intensities in theintestine of AOM injected mice as determined by measuring the numbers ofphotons collected from AOM injected mice divided by the number ofphotons collected from PBS injected mice (FIG. 9B);

FIGS. 10A-10B include graphs showing the ability of lower concentrationsof curcumin encapsulated in the GELPS to not induce apoptosis of humanmonocytes and to suppress T cell proliferation, including: a graphshowing the results of FACS analysis of CD11b+PI+AnnexinV+ cells (FIG.10A); and a graph showing the extent of T cell proliferation as wasmeasured by ³H- thymidine incorporation in GELP-Cur treated mice (FIG.10B);

FIG. 11 includes a schematic diagram showing a model used to testwhether oral GELP-Cur treatment of rheumatoid arthritis patients is safeand leads to selective induction of monocyte apoptosis and subsequentattenuation of RA progression, and to test whether monocytes taking upless GELP-Cur nanoparticles result in the accumulation of curcumin orits metabolic products in inflamed joints and further inhibit theproliferation of other infiltrating immune cells;

FIGS. 12A-12D include images and graphs showing the transfectionefficiency of grapefruit liposome as a nucleic acid delivery vehicle,including: images showing the expression of green fluorescent protein(GFP) in B6 spleen T cells using the transfection agents Fusion HD,LIPOFECTAMINE® 2000 (Invitrogen, Carlsbad, CA), and grapefruit liposome(FIG. 12A); graphs showing representative FACS of GFP positive cells(FIG. 12B), and graphs showing the transfection efficiency percentages(FIG. 12C) or the percent of transfected and untransfected cells (FIG.12D) as a function of the various delivery vehicles;

FIGS. 13A-13B are graphs showing the ability of grapefruit liposomes totransfect a broad spectrum of cell lines, including: a graph showing thetransfection efficiency of the transfection agents, Fusion HD andLIPOFECTAMINE® 2000 (Invitrogen, Carlsbad, CA), and grapefruit liposomeas measured by FACS analysis for GFP⁺ cells (FIG. 13A); and a graphshowing the percent of cytoxocity in the transfected cells as measuredby the extent of propidium iodide positive cells (FIG. 13B);

FIG. 14 is a graph showing the ability of grapefruit liposome toeffectively deliver a siRNA, where GL26-Luc microglioma cells stablyexpressing a luciferase gene were transfected with either luciferasesiRNA or scrambled siRNA (1 µg/well of a 6-well plate) using thetransfection agents Fusion HD and LIPOFECTAMINE® 2000 (Invitrogen,Carlsbad, CA), or with grapefruit liposome;

FIGS. 15A-15E include images and graphs showing the characterization offlower-like, nano-sized particles made from grapefruit-derived lipids,including: an image showing sucrose banded particles from grapefruitjuice (FIG. 15A, left) and electromicroscopy visualization of nano-sizedparticles at low and high magnification (FIG. 15A, middle and right);images showing sucrose banded grapefruit lipid-derived edible plantnano-vectors (FIG. 15B, EPNVs, left) and electromicroscopy visualizationof the nano-sized particles at low and high magnification (FIG. 15B,middle and right); a pie chart with a summary of the putative lipidspecies in the EPNVs (FIG. 15C); images showing electromicroscopyanalysis of EPNVs before (left) and after (right) homogenization (FIG.15D); and images showing electromicroscopy analysis of EPNVs embedded inpolyBed 812, sectioned, and examined by electromicroscopy (FIG. 15E);

FIGS. 16A-16F include images and graphs showing the ability of bothhematopoietic and non-hematopoietic cells to take up EPNVs; including:confocal images (FIG. 16A) of EPNVs taken up by various tumor cells(A549, GL26, 4T1, SW620 and CT26) and primary lymphocytes (T and Bcells); graphs showing the FACS quantitative analysis of uptakeefficiency of EPNVs (FIG. 16B); graphs showing the temperature (T, FIG.16C), Time (FIG. 16D) and concentration dependence (FIG. 16E) of theefficiency of EPNVs uptake; and graphs showing the identification ofpotential pathways utilized by EPNVs to enter A549 cells (FIG. 16F);.

FIGS. 17A-17G are images and graphs showing the in vitro and in vivostability and biodistribution of EPNVs, including: an image and graphsshowing the stability of PKH26-labeled EPNVs and curcumin-loaded EPNVsin PBS as indicated by color changes of PKH26 labeled EPNVs (FIG. 17A,left) and anti-inflammatory effects of curcumin loaded EPNVs oninhibition of LPS induced inflammatory cytokines (FIG. 17A, right);images and a graph showing the biodistribution of DiR dye-labeled EPNVsin mice either administered subcutaneously (s.c.), intraperitoneally(i.p.), intravenously (i.v.), and intramuscularly (i.m.) (FIG. 17B); animage and a graph showing the tissue biodistribution over time of DiRdye-labeled EPNVs administrated i.v. to mice (FIG. 17C); graphs showingin vivo cell targeting as determined by FACS analysis of totalsplenocytes and liver cells of mice injected i.v. with PKH26 labeledEPNV (FIG. 17D); images and a graph showing in vivo stability of DiRdye-labeled EPNVs as determined by scanning various organs of miceinjected i.v. with DiR dye labeled EPNVs (FIG. 17E); and images and agraph showing the in vivo stability of circulating EPNVs as determinedby scanning peripheral blood of mice injected i.v. with DiR dye-labeledEPNVs (FIG. 17F); and images of pregnant mice injected i.v. with DiRdye-labeled EPNVs either 1 (1x, left) or 5 times (5x, right), where theDiR signals are indicated by an arrow in the fetus and placenta (circleddotted line) (FIG. 17G);

FIGS. 18A-18C are images and graphs showing EPNV delivery ofbiotinylated DNA expression vectors, antibodies and siRNA, including:images and graphs showing A549 cells transfected with biotinylated eYFPcarried by EPNVs or LIPOFECTAMINE® 2000 (Invitrogen, Carlsbad, CA) whereYFP positive cells were quantitatively analyzed by FACS (FIG. 18A);graphs showing the quantitative analysis of PKH26 positive cells, wherePKH26- EPNVs loaded with biotin labeled anti-CD4 or anti-CD8 antibodieswere incubated with splenocytes in vitro (FIG. 18B); and graphs showingthe biological effect of luciferase specific siRNA carried by EPNVs orLIPOFECTAMINE® 2000 (Invitrogen, Carlsbad, CA) on inhibition ofluciferase activity of transfected GL-26-luc and A549-luc cells (FIG.18C);

FIGS. 19A-19E include graphs and images showing the ability of EPNVs tocarry anti-cancer therapeutic agents using a targeting moiety to tumortissue, including: images and graphs showing the results of experimentswhere C57BL/6J mice were implanted with GL26-Luc cells, EPNVs wereloaded with Stat3 inhibitor JSI-124 and then intranasally administratedto mice, and where the mice were imaged on post-injection days asindicated, the growth potential of injected GL26-Luc cells wasdetermined by dividing photon emissions of mice treated with PBS by thephoton emissions of mice treated with EPNV, JSI124, or EPNV-JSI124 , andwhere the percent of EPNV-JSI124-treated mice surviving was compared tocontrol mice (FIG. 19A); images and graphs showing EPNV-mediatedtargeting co-delivery of PTX and folic acid to mouse CT26, human SW620colon tumors and the mouse 4T1 breast tumor (FIG. 19B); and graphsshowing the tumor volume in mice subsequent to the injection of tumorcells into BALB/c mice (CT26, and 4T1, 1x10⁶/mouse) or NOD-SCID mice(SW620, 5x10⁶/mouse) (FIG. 19C); images and graphs showing the tumorsand DiR fluorescent signals of the tumor 30 days post-implantation (FIG.19D); images and graphs showing EPNV-mediated in vivo delivery of siRNAto tumors, where CT26-Luc tumor cell-bearing mice were intravenouslyinjected with luciferase siRNA (50 pmol/mouse in 200 nmol EPNVs),luciferase siRNA carried by EPNVs, or folic acid and luciferase siRNAco-delivered by EPNVs (FIG. 19E);

FIG. 20 includes images showing particles reassembled from lipidsderived from two other bands of sucrose purified grapefruit particles;

FIG. 21 includes images showing the characterization of signals forPKH26 labeled EPNVs or free PKH26 dye taken up by A549 cells, where theA549 cells were incubated with free PKH26 dye (top) or PKH26 labeledEPNVs (bottom), and the pattern of PKH26+ dots was imaged using confocalmicroscope;

FIG. 22 includes graphs showing the effects of metabolicinhibitor-sodium azide and pH values on EPNVs uptake, where A549 cellswere cultured with PKH26 labeled EPNVs for 3 or 6 hours in presence of50 mM sodium azide, then the percentage of taking up was qualitativelyanalyzed using FACS;

FIG. 23 includes further graphs showing the effects of pH values onEPNVs uptake, where A549 cells were cultured with PKH26 labeled EPNVs inDMEM media with different pH values (5.5, 6.5, 7.4 and 9.0) for 6 hours,and uptake efficiency was evaluated by FACS analysis;

FIGS. 24A- 24B includes graphs showing the effect of EPNVs on cellproliferation and apoptosis, including: a graph showing the analysis ofthe proliferation of EPNV-treated A549 cells using an ATPlite assay 24,48 or 72 h after exposure to different doses of the EPNV (FIG. 24A); andgraphs showing the extent of apoptosis in EPNV-treated A549 cells asanalyzed by FACS analysis of Annexin V+/PI+ cells (FIG. 24B);

FIGS. 25A-25C include graphs and images showing the in vivo cytotoxicityanalyses of EPNVs, including: a graph showing the sera levels AST andALT liver enzymes in female C57BL/6j mice (n=5) injected i.v. with 100nmol EPNV 1 (1x) or 5 times (5x) (FIG. 25A); graphs showing the levelsof inflammatory cytokines in the mice (FIG. 25B); and haematoxilin andeosin stained sections of livers, spleens, kidneys, and lungs fromEPNV-treated mice (FIG. 25C);

FIG. 26 includes images and graphs showing the ability of EPNVs to notaffect the biological functions of agents carried by EPNVs, where theanti-inflammatory agent curcumin (top, left), folic acid (FA), a ligandof folate receptor (medium, left) and an immune stimulator, β-glucan(bottom, left) were loaded onto EPNVs and isolated on a sucrosegradient, and where curcumin mediated inhibition of induction of TNF-αand IL-6 produced by LPS (100 ng/ml) stimulated mouse splenocytes (top,right), FA mediated uptake of EPNVs by CT26 and SW620 cells (medium,right), and production of TNF-α and IFN-γ by β-glucan stimulated mousesplenocytes (bottom, right) was measured;

FIG. 27 includes an image and a graph showing the ability of EPNVs tocarry a biotin labeled eYFP vector;

FIG. 28 is an image showing a western blot analysis of the expression ofphosphorylated stat3, where A549 cells were treated with PBS, EPNV,JSI124 or EPNVs-JSI124 and the expression of p-Stat3 and Stat3 wasanalyzed by western blots;

FIG. 29 includes images and graphs showing tumor sizes of three folicacid-mediated EPNV targeting tumor models, where on day 30 afterinjecting mice with tumor cells, the mice were sacrificed and CT26tumors (top), SW620 tumors (medium) and 4T1 (bottom) were removed,photographed (left) and the size measured (right); and

FIGS. 30A-30C include images showing the biodistribution of EPNV,EPNV-FA, EPNV-PTX and EPNV-FA-PTX, where on day 30 after injecting micewith tumor cells, mice were sacrificed, the organs (spleen, liver,kidney, brain, lung, heart, thymus and gut) removed and the distributionof DIR dye labeled EPNVs was images and the signals quantified.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document. The information provided in thisdocument, and particularly the specific details of the describedexemplary embodiments, is provided primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom. In case of conflict, the specification of this document,including definitions, will control.

While the terms used herein are believed to be well understood by one ofordinary skill in the art, definitions are set forth herein tofacilitate explanation of the presently-disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently-disclosed subject matter belongs.Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently-disclosed subject matter, representative methods, devices, andmaterials are now described.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a cell” includes aplurality of such cells, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about”. Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

As used herein, ranges can be expressed as from “about” one particularvalue, and/or to “about” another particular value. It is also understoodthat there are a number of values disclosed herein, and that each valueis also herein disclosed as “about” that particular value in addition tothe value itself. For example, if the value “10” is disclosed, then“about 10” is also disclosed. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

Microvesicles are naturally existing nanoparticles that are in the formof small assemblies of lipid particles, are about 50 to 1000 nm in size,and are not only secreted by many types of in vitro cell cultures and invivo cells, but are commonly found in vivo in body fluids, such asblood, urine and malignant ascites. Indeed, microvesicles include, butare not limited to, particles such as exosomes, epididimosomes,argosomes, exosome-like vesicles, microparticles, promininosomes,prostasomes, dexosomes, texosomes, dex, tex, archeosomes, and oncosomes.

As noted above, microvesicles can be formed by a variety of processes,including the release of apoptotic bodies, the budding of microvesiclesdirectly from the cytoplasmic membrane of a cell, and exocytosis frommultivesicular bodies. For example, exosomes are commonly formed bytheir secretion from the endosomal membrane compartments of cells as aconsequence of the fusion of multivesicular bodies with the plasmamembrane. The multivesicular bodies are formed by inward budding fromthe endosomal membrane and subsequent pinching off of small vesiclesinto the luminal space. The internal vesicles present in the MVBs arethen released into the extracellular fluid as so-called exosomes.

As part of the formation and release of microvesicles, unwantedmolecules are eliminated from cells. However, cytosolic and plasmamembrane proteins are also incorporated during these processes into themicrovesicles, resulting in microvesicles having particle sizeproperties, lipid bilayer functional properties, and other uniquefunctional properties that allow the microvesicles to potentiallyfunction as effective nanoparticle carriers of therapeutic agents. Inthis regard, the term “microvesicle” is used interchangeably herein withthe terms “nanoparticle,” “liposome,” “exosome,” “exosome-likeparticle,” “nano-vector” and grammatical variations of each of theforegoing. It has now been discovered though that edible plants, such asfruits, are not only a viable source of large quantities ofmicrovesicles, but that microvesicles derived from edible plants can beused as a effective delivery vehicle for a number of therapeutic agents,while also retaining the biological activity of the therapeutic agents.

The presently-disclosed subject matter thus includes edibleplant-derived microvesicle compositions that further include therapeuticagents and are useful in the treatment of various diseases, includinginflammatory disorders and cancers. In some embodiments of thepresently-disclosed subject matter, a microvesicle composition isprovided that comprises a therapeutic agent encapsulated by anmicrovesicle, wherein the microvesicle is derived from an edible plant.In some embodiments, the therapeutic agent encapsulated by theedible-plant derived microvesicle is selected from a phytochemicalagent, a stat3 inhibitor, and a chemotherapeutic agent.

The term “edible plant” is used herein to describe organisms from thekingdom Plantae that are capable of producing their own food, at leastin part, from inorganic matter through photosynthesis, and that are fitfor consumption by a subject, as defined herein below. Such edibleplants include, but are not limited to, vegetables, fruits, nuts, andthe like. In some embodiments of the microvesicle compositions describedherein, the edible plant is a fruit. In some embodiments, the fruit isselected from a grape, a grapefruit, and a tomato.

The phrase “derived from an edible plant,” when used in the context of amicrovesicle derived from an edible plant, refers to a microvesiclethat, by the hand of man, exists apart from its native environment andis therefore not a product of nature. In this regard, in someembodiments, the phrase “derived from an edible plant” can be usedinterchangeably with the phrase “isolated from an edible plant” todescribe a microvesicle of the presently-disclosed subject matter thatis useful for encapsulating therapeutic agents.

The phrase “encapsulated by a microvesicle,” or grammatical variationsthereof is used herein to refer to microvesicles whose lipid bilayersurrounds a therapeutic agent. For example, a reference to “microvesiclecurcumin” refers to an microvesicle whose lipid bilayer encapsulates orsurrounds an effective amount of curcumin. In some embodiments, theencapsulation of various therapeutic agents within microvesicles can beachieved by first mixing the one or more of the phytochemical agents,Stat3 inhibitors, or chemotherapeutic agents with isolated microvesiclesin a suitable buffered solution, such as phosphate-buffered saline(PBS). After a period of incubation sufficient to allow the therapeuticagent to become encapsulated during the incubation period, themicrovesicle/therapeutic agent mixture is then subjected to a sucrosegradient (e.g., and 8, 30, 45, and 60% sucrose gradient) to separate thefree therapeutic agent and free microvesicles from the therapeuticagents encapsulated within the microvesicles, and a centrifugation stepto isolate the microvesicles encapsulating the therapeutic agents. Afterthis centrifugation step, the microvesicles including the therapeuticagents are seen as a band in the sucrose gradient such that they canthen be collected, washed, and dissolved in a suitable solution for useas described herein below.

As noted, in some embodiments, the therapeutic agent is a phytochemicalagent. As used herein, the term “phytochemical agent” refers to anon-nutritive plant-derived compound, or an analog thereof. Examples ofphytochemical agents include, but are not limited to compounds such asmonophenols; flavonoids, such as flavonols, flavanones, flavones,flavan-3-ols, anthocyanins, anthocyanidins, isoflavones,dihydroflavonols, chalcones, and coumestans; phenolic acids;hydroxycinnamic acids; lignans; tyrosol esters; stillbenoids;hydrolysable tannins; carotenoids, such as carotenes and xanthophylls;monoterpenes; saponins; lipids, such as phytosterols, tocopherols, andomega-3,6,9 fatty acids; diterpenes; triterpinoids; betalains, such asbetacyanins and betaxanthins; dithiolthiones; thiosulphonates; indoles;and glucosinolates. As another example of a phytochemical agentdisclosed herein, the phytochemical agent can be an analog of aplant-derived compound, such as oltipraz, which is an analog of1,2-dithiol-3-thione, a compound that is found in many cruciferousvegetables.

In some embodiments of the presently-disclosed subject matter, thetherapeutic agent is a phytochemical agent selected from curcumin,resveratrol, baicalein, fisetin, and quercetin. In some embodiments, thephytochemical agent is curcumin. Curcumin is a pleiotropic naturalpolyphenol with anti-inflammatory, anti-neoplastic, anti-oxidant andchemopreventive activity, with these activities having been identifiedat both the protein and molecular levels. Nevertheless, limited progresshas been reported with respect to the therapeutic use of curcumin ascurcumin is insoluble in aqueous solvents and is relatively unstable. Inaddition, curcumin is known to have a low systemic bioavailability afteroral dosing, which further limits its usage and clinical efficacy. Ithas been determined, however, that by encapsulating curcumin in edibleplant derived microvesicle, not only can the solubility of curcumin beincreased, but the encapsulation of the curcumin within themicrovesicles protects the curcumin from degradation and also increasesthe bioavailability of the microvesicle curcumin.

As also noted herein above, in some embodiments of thepresently-disclosed subject matter, the therapeutic agent that isencapsulated within the exosome is a chemotherapeutic agent. Examples ofchemotherapeutic agents that can be used in accordance with thepresently-disclosed subject matter include, but are not limited to,platinum coordination compounds such as cisplatin, carboplatin oroxalyplatin; taxane compounds, such as paclitaxel or docetaxel;topoisomerase I inhibitors such as camptothecin compounds for exampleirinotecan or topotecan; topoisomerase II inhibitors such as anti-tumorpodophyllotoxin derivatives for example etoposide or teniposide;anti-tumor vinca alkaloids for example vinblastine, vincristine orvinorelbine; anti-tumor nucleoside derivatives for example5-fluorouracil, gemcitabine or capecitabine; alkylating agents, such asnitrogen mustard or nitrosourea for example cyclophosphamide,chlorambucil, carmustine or lomustine; anti-tumor anthracyclinederivatives for example daunorubicin, doxorubicin, idarubicin ormitoxantrone; HER2 antibodies for example trastuzumab; estrogen receptorantagonists or selective estrogen receptor modulators for exampletamoxifen, toremifene, droloxifene, faslodex or raloxifene; aromataseinhibitors, such as exemestane, anastrozole, letrazole and vorozole;differentiating agents such as retinoids, vitamin D and retinoic acidmetabolism blocking agents (RAMBA) for example accutane; DNA methyltransferase inhibitors for example azacytidine; kinase inhibitors forexample flavoperidol, imatinib mesylate or gefitinib;farnesyltransferase inhibitors; HDAC inhibitors; other inhibitors of theubiquitin-proteasome pathway for example VELCADE® (MillenniumPharmaceuticals, Cambridge, MA); or YONDELIS® (Johnson & Johnson, NewBrunswick, NJ). In some embodiments, the chemotherapeutic agent that isencapsulated by an exosome in accordance with the presently-disclosedsubject matter is selected from retinoic acid, 5-fluorouracil,vincristine, actinomycin D, adriamycin, cisplatin, docetaxel,doxorubicin, and taxol.

As further noted, in some embodiments, the therapeutic agent is a signaltransducer and activator of transcription 3 (Stat3) inhibitor. “Stat3”or “Signal Transducer and Activator of Transcription 3” is atranscription factor encoded by the STAT3 gene and, in response tocytokines or growth factors, is known to become phosphorylated and tothen translocate to the nucleus of cells where it mediates theexpression of a variety of genes in response to various stimuli, andthus plays a role in a number of cellular processes including cellgrowth and apoptosis. In this regard, the term “Stat3 inhibitor” is usedherein to refer to any chemical compound or protein that prevents orotherwise reduces the activity of Stat3 including, but not limited to,chemical compounds or proteins that prevent or reduce thetranscriptional activity of Stat3, and chemical compounds or proteinsthat prevent or reduce the activation of Stat3 by preventing itsactivation (e.g., the phosphorylation and/or translocation of Stat3 tothe nucleus of a cell). A number of Stat3 inhibitors are known to thoseskilled in the art including, but not limited to, the PIAS3 protein,Stattin, or JSI-124, which is also referred to as curcurbitacin I. Insome embodiments of the presently-disclosed subject matter, the Stat3inhibitor that is encapsulated within the edible plant-derivedmicrovesicles is JSI-124.

In other embodiments of the presently-disclosed subject matter,therapeutic agents included within the microvesicle compositionscomprises nucleic acid molecules selected from a siRNA, a microRNA, andan expression vector, such as a mammalian expression vector. The term“nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid and are metabolized in a mannersimilar to naturally occurring nucleotides. Unless otherwise indicated,a particular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences and as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions canbe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res 19:5081;Ohtsuka et al. (1985) J Biol Chem 260:2605-2608; Rossolini et al. (1994)Mol Cell Probes 8:91-98). The terms “nucleic acid” or “nucleic acidsequence” can also be used interchangeably with gene, open reading frame(ORF), cDNA, mRNA, siRNA, microRNA, and the like.

The terms “small interfering RNA,” “short interfering RNA,” “smallhairpin RNA,” “siRNA,” and “shRNA” are used interchangeably herein torefer to any nucleic acid molecule capable of mediating RNA interference(RNAi) or gene silencing. See, e.g., Bass, Nature 411:428-429, 2001;Elbashir et al., Nature 411:494-498, 2001a; and PCT InternationalPublication Nos. WO 00/44895, WO 01/36646, WO 99/32619, WO 00/01846, WO01/29058, WO 99/07409, and WO 00/44914. In one embodiment, the siRNA cancomprise a double stranded polynucleotide molecule comprisingcomplementary sense and antisense regions, wherein the antisense regioncomprises a sequence complementary to a region of a target nucleic acidmolecule. In another embodiment, the siRNA can comprise a singlestranded polynucleotide having self-complementary sense and antisenseregions, wherein the antisense region comprises a sequence complementaryto a region of a target nucleic acid molecule. In yet anotherembodiment, the siRNA can comprise a single stranded polynucleotidehaving one or more loop structures and a stem comprising selfcomplementary sense and antisense regions, wherein the antisense regioncomprises a sequence complementary to a region of a target nucleic acidmolecule, and wherein the polynucleotide can be processed either in vivoor in vitro to generate an active siRNA capable of mediating RNAi. Asused herein, siRNA molecules need not be limited to those moleculescontaining only RNA, but further encompass chemically modifiednucleotides and non-nucleotides.

MicroRNAs are naturally occurring, small non-coding RNAs that are about17 to about 25 nucleotide bases (nt) in length in their biologicallyactive form. miRNAs post-transcriptionally regulate gene expression byrepressing target mRNA translation. It is thought that miRNAs functionas negative regulators, i.e. greater amounts of a specific miRNA willcorrelate with lower levels of target gene expression. There are threeforms of miRNAs existing in vivo, primary miRNAs (pri-miRNAs), prematuremiRNAs (pre-miRNAs), and mature miRNAs. Primary miRNAs (pri-miRNAs) areexpressed as stem-loop structured transcripts of about a few hundredbases to over 1 kb. The pri-miRNA transcripts are cleaved in the nucleusby an RNase II endonuclease called Drosha that cleaves both strands ofthe stem near the base of the stem loop. Drosha cleaves the RNA duplexwith staggered cuts, leaving a 5′ phosphate and 2 nt overhang at the 3′end. The cleavage product, the premature miRNA (pre-miRNA) is about 60to about 110 nt long with a hairpin structure formed in a fold-backmanner. Pre-miRNA is transported from the nucleus to the cytoplasm byRan-GTP and Exportin-5. Pre-miRNAs are processed further in thecytoplasm by another RNase II endonuclease called Dicer. Dicerrecognizes the 5′ phosphate and 3′ overhang, and cleaves the loop off atthe stem-loop junction to form miRNA duplexes. The miRNA duplex binds tothe RNA-induced silencing complex (RISC), where the antisense strand ispreferentially degraded and the sense strand mature miRNA directs RISCto its target site. It is the mature miRNA that is the biologicallyactive form of the miRNA and is about 17 to about 25 nt in length.

In some embodiments, the nucleic acid molecules that are encapsulated orotherwise incorporated into a microvesicle composition of thepresently-disclosed subject matter are included in the microvesicles arepart of an expression vector. The term “expression vector” is usedinterchangeably herein with the terms “expression cassette” and“expression control sequence,” and is used to refer to a nucleic acidmolecule capable of directing expression of a particular nucleotidesequence in an appropriate host cell, comprising a promoter operativelylinked to the nucleotide sequence of interest which is operativelylinked to termination signals. It also typically comprises sequencesrequired for proper translation of the nucleotide sequence. The codingregion usually encodes a polypeptide of interest but can also encode afunctional RNA of interest, for example antisense RNA or anon-translated RNA, in the sense or antisense direction. The expressionvector comprising the nucleotide sequence of interest can be chimeric,meaning that at least one of its components is heterologous with respectto at least one of its other components. The expression vector can alsobe one that is naturally occurring but has been obtained in arecombinant form useful for heterologous expression. In someembodiments, the expression vector is a mammalian expression vector thatis capable of directing expression of a particular nucleic acid sequenceof interest in a mammalian cell.

In some embodiments of the presently disclosed subject matter, apharmaceutical composition is provided that comprises an edibleplant-derived microvesicle composition disclosed herein and apharmaceutical vehicle, carrier, or excipient. In some embodiments, thepharmaceutical composition is pharmaceutically-acceptable in humans.Also, as described further below, in some embodiments, thepharmaceutical composition can be formulated as a therapeuticcomposition for delivery to a subject.

A pharmaceutical composition as described herein preferably comprises acomposition that includes pharmaceutical carrier such as aqueous andnon-aqueous sterile injection solutions that can contain antioxidants,buffers, bacteriostats, bactericidal antibiotics and solutes that renderthe formulation isotonic with the bodily fluids of the intendedrecipient; and aqueous and non-aqueous sterile suspensions, which caninclude suspending agents and thickening agents. The pharmaceuticalcompositions used can take such forms as suspensions, solutions, oremulsions in oily or aqueous vehicles, and can contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.Additionally, the formulations can be presented in unit-dose ormulti-dose containers, for example sealed ampoules and vials, and can bestored in a frozen or freeze-dried or room temperature (lyophilized)condition requiring only the addition of sterile liquid carrierimmediately prior to use.

In some embodiments, solid formulations of the compositions for oraladministration can contain suitable carriers or excipients, such as cornstarch, gelatin, lactose, acacia, sucrose, microcrystalline cellulose,kaolin, mannitol, dicalcium phosphate, calcium carbonate, sodiumchloride, or alginic acid. Disintegrators that can be used include, butare not limited to, microcrystalline cellulose, corn starch, sodiumstarch glycolate, and alginic acid. Tablet binders that can be usedinclude acacia, methylcellulose, sodium carboxymethylcellulose,polyvinylpyrrolidone, hydroxypropyl methylcellulose, sucrose, starch,and ethylcellulose. Lubricants that can be used include magnesiumstearates, stearic acid, silicone fluid, talc, waxes, oils, andcolloidal silica. Further, the solid formulations can be uncoated orthey can be coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide asustained/extended action over a longer period of time. For example,glyceryl monostearate or glyceryl distearate can be employed to providea sustained-/extended-release formulation. Numerous techniques forformulating sustained release preparations are known to those ofordinary skill in the art and can be used in accordance with the presentinvention, including the techniques described in the followingreferences: U.S. Pat. Nos. 4,891,223; 6,004,582; 5,397,574; 5,419,917;5,458,005; 5,458,887; 5,458,888; 5,472,708; 6,106,862; 6,103,263;6,099,862; 6,099,859; 6,096,340; 6,077,541; 5,916,595; 5,837,379;5,834,023; 5,885,616; 5,456,921; 5,603,956; 5,512,297; 5,399,362;5,399,359; 5,399,358; 5,725,883; 5,773,025; 6,110,498; 5,952,004;5,912,013; 5,897,876; 5,824,638; 5,464,633; 5,422,123; and 4,839,177;and WO 98/47491, each of which is incorporated herein by this reference.

Liquid preparations for oral administration can take the form of, forexample, solutions, syrups, or suspensions, or they can be presented asa dry product for constitution with water or other suitable vehiclebefore use. Such liquid preparations can be prepared by conventionaltechniques with pharmaceutically-acceptable additives such as suspendingagents (e.g., sorbitol syrup, cellulose derivatives or hydrogenatededible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations can alsocontain buffer salts, flavoring, coloring, and sweetening agents asappropriate. Preparations for oral administration can be suitablyformulated to give controlled release of the active compound. For buccaladministration, the compositions can take the form of capsules, tabletsor lozenges formulated in conventional manner.

Various liquid and powder formulations can also be prepared byconventional methods for inhalation into the lungs of the subject to betreated or for intranasal administration into the nose and sinuscavities of a subject to be treated . For example, the compositions canbe conveniently delivered in the form of an aerosol spray presentationfrom pressurized packs or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas.Capsules and cartridges of, for example, gelatin for use in an inhaleror insufflator may be formulated containing a powder mix of the desiredcompound and a suitable powder base such as lactose or starch.

The compositions can also be formulated as a preparation forimplantation or injection. Thus, for example, the compositions can beformulated with suitable polymeric or hydrophobic materials (e.g., as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives (e.g., as a sparingly soluble salt).

Injectable formulations of the compositions can contain various carrierssuch as vegetable oils, dimethylacetamide, dimethylformamide, ethyllactate, ethyl carbonate, isopropyl myristate, ethanol, polyols(glycerol, propylene glycol, liquid polyethylene glycol), and the like.For intravenous injections, water soluble versions of the compositionscan be administered by the drip method, whereby a formulation includinga pharmaceutical composition of the presently-disclosed subject matterand a physiologically-acceptable excipient is infused.Physiologically-acceptable excipients can include, for example, 5%dextrose, 0.9% saline, Ringer’s solution or other suitable excipients.Intramuscular preparations, e.g., a sterile formulation of a suitablesoluble salt form of the compounds, can be dissolved and administered ina pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or5% glucose solution. A suitable insoluble form of the composition can beprepared and administered as a suspension in an aqueous base or apharmaceutically-acceptable oil base, such as an ester of a long chainfatty acid, (e.g., ethyl oleate).

In addition to the formulations described above, the microvesiclecompositions of the presently-disclosed subject matter can also beformulated as rectal compositions, such as suppositories or retentionenemas, e.g., containing conventional suppository bases such as cocoabutter or other glycerides. Further, the exosomal compositions can alsobe formulated as a depot preparation by combining the compositions withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

Further provided, in some embodiments of the presently-disclosed subjectmatter, are methods for treating an inflammatory disorder or a cancer.In some embodiments, a method for treating an inflammatory disorder isprovided that comprises administering to a subject in need thereof aneffective amount of a microvesicle composition of thepresently-disclosed subject matter, wherein the microvesicle included inthe composition is derived from an edible plant, such as a fruit.

As used herein, the terms “treatment” or “treating” relate to anytreatment of a condition of interest (e.g., an inflammatory disorder ora cancer), including but not limited to prophylactic treatment andtherapeutic treatment. As such, the terms “treatment” or “treating”include, but are not limited to: preventing a condition of interest orthe development of a condition of interest; inhibiting the progressionof a condition of interest; arresting or preventing the furtherdevelopment of a condition of interest; reducing the severity of acondition of interest; ameliorating or relieving symptoms associatedwith a condition of interest; and causing a regression of a condition ofinterest or one or more of the symptoms associated with a condition ofinterest.

As used herein, the term “inflammatory disorder” includes diseases ordisorders which are caused, at least in part, or exacerbated, byinflammation, which is generally characterized by increased blood flow,edema, activation of immune cells (e.g., proliferation, cytokineproduction, or enhanced phagocytosis), heat, redness, swelling, painand/or loss of function in the affected tissue or organ. The cause ofinflammation can be due to physical damage, chemical substances,micro-organisms, tissue necrosis, cancer, or other agents or conditions.

Inflammatory disorders include acute inflammatory disorders, chronicinflammatory disorders, and recurrent inflammatory disorders. Acuteinflammatory disorders are generally of relatively short duration, andlast for from about a few minutes to about one to two days, althoughthey can last several weeks. Characteristics of acute inflammatorydisorders include increased blood flow, exudation of fluid and plasmaproteins (edema) and emigration of leukocytes, such as neutrophils.Chronic inflammatory disorders, generally, are of longer duration, e.g.,weeks to months to years or longer, and are associated histologicallywith the presence of lymphocytes and macrophages and with proliferationof blood vessels and connective tissue. Recurrent inflammatory disordersinclude disorders which recur after a period of time or which haveperiodic episodes. Some inflammatory disorders fall within one or morecategories. Exemplary inflammatory disorders include, but are notlimited to atherosclerosis; arthritis; inflammation-promoted cancers;asthma; autoimmune uveitis; adoptive immune response; dermatitis;multiple sclerosis; diabetic complications; osteoporosis; Alzheimer’sdisease; cerebral malaria; hemorrhagic fever; autoimmune disorders; andinflammatory bowel disease. In some embodiments, the inflammatorydisorder is selected from the group consisting of sepsis, septic shock,colitis, colon cancer, and arthritis.

For administration of a therapeutic composition as disclosed herein(e.g., an edible plant-derived microvesicle encapsulating a therapeuticagent), conventional methods of extrapolating human dosage based ondoses administered to a murine animal model can be carried out using theconversion factor for converting the mouse dosage to human dosage: DoseHuman per kg = Dose Mouse per kg × 12 (Freireich, et al., (1966) CancerChemother Rep. 50: 219-244). Doses can also be given in milligrams persquare meter of body surface area because this method rather than bodyweight achieves a good correlation to certain metabolic and excretionaryfunctions. Moreover, body surface area can be used as a commondenominator for drug dosage in adults and children as well as indifferent animal species as described by Freireich, et al. (Freireich etal., (1966) Cancer Chemother Rep. 50:219-244). Briefly, to express amg/kg dose in any given species as the equivalent mg/sq m dose, multiplythe dose by the appropriate km factor. In an adult human, 100 mg/kg isequivalent to 100 mg/kg × 37 kg/sq m=3700 mg/m².

Suitable methods for administering a therapeutic composition inaccordance with the methods of the presently-disclosed subject matterinclude, but are not limited to, systemic administration, parenteraladministration (including intravascular, intramuscular, and/orintraarterial administration), oral delivery, buccal delivery, rectaldelivery, subcutaneous administration, intraperitoneal administration,inhalation, intratracheal installation, surgical implantation,transdermal delivery, local injection, intranasal delivery, andhyper-velocity injection/bombardment. Where applicable, continuousinfusion can enhance drug accumulation at a target site (see, e.g., U.S.Pat. No. 6,180,082).

Regardless of the route of administration, the compositions of thepresently-disclosed subject matter are typically administered in amounteffective to achieve the desired response. As such, the term “effectiveamount” is used herein to refer to an amount of the therapeuticcomposition (e.g., a microvesicle encapsulating a therapeutic agent, anda pharmaceutically vehicle, carrier, or excipient) sufficient to producea measurable biological response (e.g., a decrease in inflammation).Actual dosage levels of active ingredients in a therapeutic compositionof the present invention can be varied so as to administer an amount ofthe active compound(s) that is effective to achieve the desiredtherapeutic response for a particular subject and/or application. Ofcourse, the effective amount in any particular case will depend upon avariety of factors including the activity of the therapeuticcomposition, formulation, the route of administration, combination withother drugs or treatments, severity of the condition being treated, andthe physical condition and prior medical history of the subject beingtreated. Preferably, a minimal dose is administered, and the dose isescalated in the absence of dose-limiting toxicity to a minimallyeffective amount. Determination and adjustment of a therapeuticallyeffective dose, as well as evaluation of when and how to make suchadjustments, are known to those of ordinary skill in the art.

For additional guidance regarding formulation and dose, see U.S. Pat.Nos. 5,326,902; 5,234,933; PCT International Publication No. WO93/25521; Berkow et al., (1997) The Merck Manual of Medical Information,Home ed. Merck Research Laboratories, Whitehouse Station, New Jersey;Goodman et al., (1996) Goodman & Gilman’s the Pharmacological Basis ofTherapeutics, 9th ed. McGraw-Hill Health Professions Division, New York;Ebadi, (1998) CRC Desk Reference of Clinical Pharmacology. CRC Press,Boca Raton, Florida; Katzung, (2001) Basic & Clinical Pharmacology, 8thed. Lange Medical Books/McGraw-Hill Medical Pub. Division, New York;Remington et al., (1975) Remington’s Pharmaceutical Sciences, 15th ed.Mack Pub. Co., Easton, Pennsylvania; and Speight et al., (1997) Avery’sDrug Treatment: A Guide to the Properties, Choice, Therapeutic Use andEconomic Value of Drugs in Disease Management, 4th ed. AdisInternational, Auckland/ Philadelphia; Duch et al., (1998) Toxicol.Lett. 100-101:255-263.

In some embodiments of the therapeutic methods disclosed herein,administering an edible plant-derived microvesicle composition of thepresently-disclosed subject matter reduces an amount of an inflammatorycytokine in a subject. In some embodiments, the inflammatory cytokinecan be interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α),interferon-γ (IFN-γ), or interleukin-6 (IL-6).

Various methods known to those skilled in the art can be used todetermine a reduction in the amount of inflammatory cytokines in asubject. For example, in certain embodiments, the amounts of expressionof an inflammatory cytokine in a subject can be determined by probingfor mRNA of the gene encoding the inflammatory cytokine in a biologicalsample obtained from the subject (e.g., a tissue sample, a urine sample,a saliva sample, a blood sample, a serum sample, a plasma sample, orsub-fractions thereof) using any RNA identification assay known to thoseskilled in the art. Briefly, RNA can be extracted from the sample,amplified, converted to cDNA, labeled, and allowed to hybridize withprobes of a known sequence, such as known RNA hybridization probesimmobilized on a substrate, e.g., array, or microarray, or quantitatedby real time PCR (e.g., quantitative real-time PCR, such as availablefrom Bio-Rad Laboratories, Hercules, CA). Because the probes to whichthe nucleic acid molecules of the sample are bound are known, themolecules in the sample can be identified. In this regard, DNA probesfor one or more of the mRNAs encoded by the inflammatory genes can beimmobilized on a substrate and provided for use in practicing a methodin accordance with the presently-disclosed subject matter.

With further regard to determining levels of inflammatory cytokines insamples, mass spectrometry and/or immunoassay devices and methods canalso be used to measure the inflammatory cytokines in samples, althoughother methods can also be used and are well known to those skilled inthe art. See, e.g., U.S. Pat. Nos. 6,143,576; 6,113,855; 6,019,944;5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776;5,824,799; 5,679,526; 5,525,524; and 5,480,792, each of which is herebyincorporated by reference in its entirety. Immunoassay devices andmethods can utilize labeled molecules in various sandwich, competitive,or non-competitive assay formats, to generate a signal that is relatedto the presence or amount of an analyte of interest. Additionally,certain methods and devices, such as biosensors and opticalimmunoassays, can be employed to determine the presence or amount ofanalytes without the need for a labeled molecule. See, e.g., U.S. Pat.Nos. 5,631,171; and 5,955,377, each of which is hereby incorporated byreference in its entirety.

Any suitable immunoassay can be utilized, for example, enzyme-linkedimmunoassays (ELISA), radioimmunoassays (RIAs), competitive bindingassays, and the like. Specific immunological binding of the antibody tothe inflammatory molecule can be detected directly or indirectly. Directlabels include fluorescent or luminescent tags, metals, dyes,radionucleotides, and the like, attached to the antibody. Indirectlabels include various enzymes well known in the art, such as alkalinephosphatase, horseradish peroxidase and the like.

The use of immobilized antibodies or fragments thereof specific for theinflammatory molecules is also contemplated by the present invention.The antibodies can be immobilized onto a variety of solid supports, suchas magnetic or chromatographic matrix particles, the surface of an assayplate (such as microtiter wells), pieces of a solid substrate material(such as plastic, nylon, paper), and the like. An assay strip can beprepared by coating the antibody or a plurality of antibodies in anarray on a solid support. This strip can then be dipped into the testbiological sample and then processed quickly through washes anddetection steps to generate a measurable signal, such as for example acolored spot.

Mass spectrometry (MS) analysis can be used, either alone or incombination with other methods (e.g., immunoassays), to determine thepresence and/or quantity of an inflammatory molecule in a subject.Exemplary MS analyses that can be used in accordance with the presentinvention include, but are not limited to: liquid chromatography-massspectrometry (LC-MS); matrix-assisted laser desorption/ionizationtime-of-flight MS analysis (MALDI-TOF-MS), such as for exampledirect-spot MALDI-TOF or liquid chromatography MALDI-TOF massspectrometry analysis; electrospray ionization MS (ESI-MS), such as forexample liquid chromatography (LC) ESI-MS; and surface enhanced laserdesorption/ionization time-of-flight mass spectrometry analysis(SELDI-TOF-MS). Each of these types of MS analysis can be accomplishedusing commercially-available spectrometers, such as, for example, triplequadropole mass spectrometers. Methods for utilizing MS analysis todetect the presence and quantity of peptides, such as inflammatorycytokines, in biological samples are known in the art. See, e.g., U.S.Pats. 6,925,389; 6,989,100; and 6,890,763 for further guidance, each ofwhich are incorporated herein by this reference.

With still further regard to the various therapeutic methods describedherein, although certain embodiments of the methods disclosed hereinonly call for a qualitative assessment (e.g., the presence or absence ofthe expression of an inflammatory cytokine in a subject), otherembodiments of the methods call for a quantitative assessment (e.g., anamount of increase in the level of an inflammatory cytokine in asubject). Such quantitative assessments can be made, for example, usingone of the above mentioned methods, as will be understood by thoseskilled in the art.

The skilled artisan will also understand that measuring a reduction inthe amount of a certain feature (e.g., cytokine levels) or animprovement in a certain feature (e.g., inflammation) in a subject is astatistical analysis. For example, a reduction in an amount ofinflammatory cytokines in a subject can be compared to control level ofinflammatory cytokines, and an amount of inflammatory cytokines of lessthan or equal to the control level can be indicative of a reduction inthe amount of inflammatory cytokines, as evidenced by a level ofstatistical significance. Statistical significance is often determinedby comparing two or more populations, and determining a confidenceinterval and/or a p value. See, e.g., Dowdy and Wearden, Statistics forResearch, John Wiley & Sons, New York, 1983, incorporated herein byreference in its entirety. Preferred confidence intervals of the presentsubject matter are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%,while preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001,and 0.0001.

In some embodiments of the presently-disclosed methods for treating aninflammatory disorder described herein, the inflammatory disorder iscolitis. In some embodiments, administering the composition to a subjectincreases an amount of intestinal epithelial cell proliferation in thesubject to thereby treat the colitis. In some embodiments, administeringan effective amount of the edible plant-derived microvesiclecompositions increases an amount of Wnt-β-catenin signaling in theintestine of a subject to thereby treat the colitis.

Still further provided, in some embodiments, are methods for treating acancer. In some embodiments, a method for treating a cancer is providedthat comprises administering to a subject in need thereof an effectiveamount of an edible-plant derived microvesicle composition of thepresently-disclosed subject matter (i.e., where a microvesicleencapsulates a therapeutic agent). In some embodiments, the therapeuticagent encapsulated within the microvesicle and used to treat the canceris selected from a phytochemical agent, a chemotherapeutic agent, and aStat3 inhibitor as such agents have been found to be particularly usefulin the treatment of cancer. As used herein, the term “cancer” refers toall types of cancer or neoplasm or malignant tumors found in animals,including leukemias, carcinomas, melanoma, and sarcomas.

By “leukemia” is meant broadly progressive, malignant diseases of theblood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Leukemia diseases include, for example, acutenonlymphocytic leukemia, chronic lymphocytic leukemia, acutegranulocytic leukemia, chronic granulocytic leukemia, acutepromyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovineleukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross’ leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cellleukemia, Schilling’s leukemia, stem cell leukemia, subleukemicleukemia, and undifferentiated cell leukemia.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas include, for example, acinarcarcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cysticcarcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolarcarcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinomabasocellulare, basaloid carcinoma, basosquamous cell carcinoma,bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogeniccarcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorioniccarcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma,cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum,cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma,carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoidcarcinoma, carcinoma epitheliale adenoides, exophytic carcinoma,carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma,gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare,glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma,hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma,hyaline carcinoma, hypemephroid carcinoma, infantile embryonalcarcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelialcarcinoma, Krompecher’s carcinoma, Kulchitzky-cell carcinoma, large-cellcarcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatouscarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, signet-ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cellcarcinoma, carcinoma spongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telangiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas include, for example, chondrosarcoma, fibrosarcoma,lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy’ssarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma,ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilns’ tumor sarcoma, endometrial sarcoma,stromal sarcoma, Ewing’s sarcoma, fascial sarcoma, fibroblastic sarcoma,giant cell sarcoma, granulocytic sarcoma, Hodgkin’s sarcoma, idiopathicmultiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of Bcells, lymphoma, immunoblastic sarcoma of T-cells, Jensen’s sarcoma,Kaposi’s sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma,malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocyticsarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, andtelangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas include, forexample, acral-lentiginous melanoma, amelanotic melanoma, benignjuvenile melanoma, Cloudman’s melanoma, S91 melanoma, Harding-Passeymelanoma, juvenile melanoma, lentigo maligna melanoma, malignantmelanoma, nodular melanoma subungal melanoma, and superficial spreadingmelanoma.

Additional cancers include, for example, Hodgkin’s Disease,Non-Hodgkin’s Lymphoma, multiple myeloma, neuroblastoma, breast cancer,ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis,primary macroglobulinemia, small-cell lung tumors, primary brain tumors,stomach cancer, colon cancer, malignant pancreatic insulanoma, malignantcarcinoid, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tractcancer, malignant hypercalcemia, cervical cancer, endometrial cancer,and adrenal cortical cancer. In some embodiments, the cancer is selectedfrom the group consisting of skin cancer, head and neck cancer, coloncancer, breast cancer, brain cancer, and lung cancer. In some particularembodiments, the cancer is a brain cancer such as, in some embodiments,a glioma.

In some embodiments, the edible plant-derived microvesicle compositionsused to treat the cancer further comprise a cancer targeting moiety or,in other words, a moiety that is capable of preferentially directing acomposition of the presently-disclosed subject matter to a cancer cell.Such cancer targeting moieties include, but are not limited to, smallmolecules, proteins, or other agents that preferentially bind to cancercells. For example, in some embodiments, the cancer targeting moiety canbe an antibody that specifically binds to an epitope found predominantlyor exclusively on a cancer cell. As another example, in someembodiments, the cancer targeting moiety is folic acid, as folic acid orfolate receptors have been found to be overexpressed on a variety ofdifferent types of cancer.

In yet further embodiments of the presently-disclosed subject matter,methods for diagnosing a colon cancer in a subject are provided. In someembodiments, a method for diagnosing a colon cancer in a subject isprovided that comprises the steps of: administering to a subject aneffective amount of a microvesicle composition including an edibleplant-derived microvesicle incorporating a detectable label; determiningan amount of the detectable label in a colon of the subject; andcomparing the amount of the detectable label, if present, to a controllevel of the detectable label, wherein the subject is diagnosed ashaving colon cancer or a risk thereof if there is a measurabledifference in the amount of the detectable label as compared to thecontrol level. In some embodiments, the subject has colon cancer.

The terms “diagnosing” and “diagnosis” as used herein refer to methodsby which the skilled artisan can estimate and even determine whether ornot a subject is suffering from a given disease or condition (e.g.,colon cancer). The skilled artisan often makes a diagnosis on the basisof one or more diagnostic indicators, such as for example an amount of adetectable label attached to an exosomal composition, the amount(including presence or absence) of which is indicative of the presence,severity, or absence of the condition.

Along with diagnosis, clinical disease prognosis is also an area ofgreat concern and interest. It is important to know the stage andrapidity of advancement of the colon cancer in order to plan the mosteffective therapy. If a more accurate prognosis can be made, appropriatetherapy, and in some instances less severe therapy for the subject canbe chosen. Measurement of amounts of detectable labels attached to anedible plant-derived microvesicle composition of the presently-disclosedsubject matter can be useful in order to categorize subjects accordingto advancement of colon cancer who will benefit from particulartherapies and differentiate from other subjects where alternative oradditional therapies can be more appropriate.

As such, “making a diagnosis” or “diagnosing”, as used herein, isfurther inclusive of determining a prognosis, which can provide forpredicting a clinical outcome (with or without medical treatment),selecting an appropriate treatment (or whether treatment would beeffective), or monitoring a current treatment and potentially changingthe treatment, based on the measured amounts of detectable labelsdisclosed herein.

The phrase “determining a prognosis” as used herein refers to methods bywhich the skilled artisan can predict the course or outcome of acondition in a subject. The term “prognosis” does not refer to theability to predict the course or outcome of a condition with 100%accuracy, or even that a given course or outcome is predictably more orless likely to occur based on the presence, absence or levels of thedetectable labels attached to the exosomal compositions. Instead, theskilled artisan will understand that the term “prognosis” refers to anincreased probability that a certain course or outcome will occur; thatis, that a course or outcome is more likely to occur in a subjectexhibiting a given condition, when compared to those individuals notexhibiting the condition. For example, in individuals not exhibiting thecondition, the chance of a given outcome may be about 3%. In certainembodiments, a prognosis is about a 5% chance of a given outcome, abouta 7% chance, about a 10% chance, about a 12% chance, about a 15% chance,about a 20% chance, about a 25% chance, about a 30% chance, about a 40%chance, about a 50% chance, about a 60% chance, about a 75% chance,about a 90% chance, or about a 95% chance.

The skilled artisan will understand that associating a prognosticindicator with a predisposition to an adverse outcome is a statisticalanalysis. For example, an amount of the detectable label of greater thana control level in some embodiments can signal that a subject is morelikely to suffer from a colon cancer than subjects with a level lessthan or equal to the control level, as determined by a level ofstatistical significance. Additionally, a change in detectable labelconcentration from baseline levels can be reflective of subjectprognosis, and the degree of change in detectable label levels can berelated to the severity of adverse events.

In other embodiments, a threshold degree of change in the amounts of adetectable label can be established, and the degree of change in theamounts can simply be compared to the threshold degree of change in thelevel. A preferred threshold change in the detectable labels of thepresently-disclosed subject matter is about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 50%, about 75%, about 100%, andabout 150%. In yet other embodiments, a “nomogram” can be established,by which a level of a prognostic or diagnostic indicator can be directlyrelated to an associated disposition towards a given outcome. Theskilled artisan is acquainted with the use of such nomograms to relatetwo numeric values with the understanding that the uncertainty in thismeasurement is the same as the uncertainty in the marker concentrationbecause individual sample measurements are referenced, not populationaverages.

In some embodiments of the presently disclosed subject matter, multipledetermination of one or more diagnostic or prognostic levels of thedetectable labels attached to the administered exosomal compositions canbe made, and a temporal change in the label levels can be used tomonitor the progression of disease and/or efficacy of appropriatetherapies directed against the disease. In such an embodiment forexample, one might expect to see a decrease or an increase in the labelsover time during the course of effective therapy. Thus, thepresently-disclosed subject matter provides, in some embodiments, amethod for determining treatment efficacy and/or progression of a coloncancer in a subject. In some embodiments, the method comprisesadministering the edible plant-derived microvesicle compositionsincluding a detectable label at various time points and determining anamount of the detectable labels in the subject at a plurality ofdifferent time points such that those amounts can be compared with oneanother to provide an assessment of the measurements collected at thedifferent time points. For example, a first time point can be selectedprior to initiation of a treatment and a second time point can beselected at some time after initiation of the treatment. One or morelevels of the detectable labels can then be measured at each of thedifferent time points and qualitative and/or quantitative differencesnoted. A change in the amounts of the levels from the first and secondsamples can be correlated with determining treatment efficacy and/orprogression of the disease in the subject.

The terms “correlated” and “correlating,” as used herein, refers tocomparing the presence or quantity of the measured detectable label in asubject to its presence or quantity in subjects known to suffer from, orknown to be at risk of, a given condition (e.g., a colon cancer); or insubjects known to be free of a given condition, i.e. “normalindividuals”. For example, a level in a particular subject can becompared to a level known to be associated with a specific type of coloncancer. The subject’s measured level of the detectable label subsequentto the administration of the labeled exosomal compositions is said tohave been correlated with a diagnosis; that is, the skilled artisan canuse the level to determine whether the subject suffers from a specifictype of colon cancer, and respond accordingly. Alternatively, thesubject’s level can be compared to a control level known to beassociated with a good outcome (e.g., the absence of colon cancer), suchas an average level found in a population of normal subjects.

As noted, in some embodiments, multiple determination of one or moreamounts of detectable labels can be made, and a temporal change in theamounts can be used to determine a diagnosis or prognosis. For example,a diagnostic level of the labels can be determined at an initial time,and again at a second time. In such embodiments, an increase in theamounts from the initial time to the second time can be diagnostic of aparticular type of colon cancer, or a given prognosis. Likewise, adecrease in the levels from the initial time to the second time can beindicative of a particular type of colon cancer or a given prognosis.Furthermore, the degree of change can be related to the severity ofcolon cancer and future adverse events.

With regard to the term “detectable label,” as used herein, the terms“detectable label,” “label” and “labeled” refer to the attachment orincorporation (e.g., encapsulation) of a moiety, capable of detection byspectroscopic, radiologic, or other methods, to a microvesicle. Examplesof labels include, but are not limited to, the following: radioisotopes,fluorescent labels, heavy atoms, enzymatic labels or reporter genes,chemiluminescent groups, biotinyl groups, predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for antibodies, metal binding domains, epitopetags). In some embodiments, labels are attached by spacer arms ofvarious lengths to reduce potential steric hindrance. In someembodiments, the detectable label comprises a radioisotope or afluorescent probe.

Fluorescent probes that can be utilized include, but are not limited tofluorescein isothiocyanate; fluorescein dichlorotriazine and fluorinatedanalogs of fluorescein; naphthofluorescein carboxylic acid and itssuccinimidyl ester; carboxyrhodamine 6G; pyridyloxazole derivatives;Cy2, 3, 3.5, 5, 5.5, and 7; phycoerythrin; phycoerythrin-Cy conjugates;fluorescent species of succinimidyl esters, carboxylic acids,isothiocyanates, sulfonyl chlorides, and dansyl chlorides, includingpropionic acid succinimidyl esters, and pentanoic acid succinimidylesters; succinimidyl esters of carboxytetramethylrhodamine; rhodamineRed-X succinimidyl ester; Texas Red sulfonyl chloride; Texas Red-Xsuccinimidyl ester; Texas Red-X sodium tetrafluorophenol ester; Red-X;Texas Red dyes; tetramethylrhodamine; lissamine rhodamine B;tetramethylrhodamine; tetramethylrhodamine isothiocyanate;naphthofluoresceins; coumarin derivatives (e.g., hydroxycoumarin,aminocoumarin, and methoxycoumarin); pyrenes; pyridyloxazolederivatives; dapoxyl dyes; Cascade Blue and Yellow dyes; benzofuranisothiocyanates; sodium tetrafluorophenols;4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; Alexa fluors (e.g., 350,430,488, 532, 546, 555, 568, 594, 633, 647, 660, 680, 700, and 750);green fluorescent protein; and yellow fluorescent protein. The peakexcitation and emission wavelengths will vary for these compounds andselection of a particular fluorescent probe for a particular applicationcan be made in part based on excitation and/or emission wavelengths.

As used herein, the term “subject” includes both human and animalsubjects. Thus, veterinary therapeutic uses are provided in accordancewith the presently disclosed subject matter. As such, thepresently-disclosed subject matter provides for the treatment of mammalssuch as humans, as well as those mammals of importance due to beingendangered, such as Siberian tigers; of economic importance, such asanimals raised on farms for consumption by humans; and/or animals ofsocial importance to humans, such as animals kept as pets or in zoos.Examples of such animals include but are not limited to: carnivores suchas cats and dogs; swine, including pigs, hogs, and wild boars; ruminantsand/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats,bison, and camels; and horses. Also provided is the treatment of birds,including the treatment of those kinds of birds that are endangeredand/or kept in zoos, as well as fowl, and more particularly domesticatedfowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guineafowl, and the like, as they are also of economic importance to humans.Thus, also provided is the treatment of livestock, including, but notlimited to, domesticated swine, ruminants, ungulates, horses (includingracehorses), poultry, and the like.

The practice of the presently-disclosed subject matter can employ,unless otherwise indicated, conventional techniques of cell biology,cell culture, molecular biology, transgenic biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See e.g.,Molecular Cloning A Laboratory Manual (1989), 2nd Ed., ed. by Sambrook,Fritsch and Maniatis, eds., Cold Spring Harbor Laboratory Press,Chapters 16 and 17; U.S. Pat. No. 4,683,195; DNA Cloning, Volumes I andII, Glover, ed., 1985; Oligonucleotide Synthesis, M. J. Gait, ed., 1984;Nucleic Acid Hybridization, D. Hames & S. J. Higgins, eds., 1984;Transcription and Translation, B. D. Hames & S. J. Higgins, eds., 1984;Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., 1987;Immobilized Cells And Enzymes, IRL Press, 1986; Perbal (1984), APractical Guide To Molecular Cloning; See Methods In Enzymology(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells,J. H. Miller and M. P. Calos, eds., Cold Spring Harbor Laboratory, 1987;Methods In Enzymology, Vols. 154 and 155, Wu et al., eds., AcademicPress Inc., N.Y.; Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987; Handbook OfExperimental Immunology, Volumes I-IV, D. M. Weir and C. C. Blackwell,eds., 1986.

The presently-disclosed subject matter is further illustrated by thefollowing specific but non-limiting examples. Some of the followingexamples are prophetic, notwithstanding the numerical values, resultsand/or data referred to and contained in the examples. Additionally, thefollowing examples may include compilations of data that arerepresentative of data gathered at various times during the course ofdevelopment and experimentation related to the presently-disclosedsubject matter.

EXAMPLES

Throughout the following examples, the terms “exosome,” “exosome-likeparticles,” “ELP,” “nano-vector,” “edible plant-derived nano-vector,”“EPNV,” and grammatical variations thereof and other similar terms areused interchangeably to refer to the microvesicle compositions of thepresently-disclosed subject matter, as described herein above.

Materials and Methods for Examples 1-6

Purification of exosomes from grapefruit and grapes. Fresh grapefruitand grapes were purchased from a local market and were washed andsterilized under a UV lamp for 30 min and processed using GoodLaboratory Practice (GLP) procedures. The fruit was cut into two pieces,and the fruit fluids were collected into centrifuge tubes for exosomalpurification using gradient centrifugation. Briefly, the skin of thefruit was removed by centrifugation for 10 min at 200 x g. Supernatantswere collected and centrifuged sequentially twice for 10 min at 500 xg_(max), once for 15 min at 2,000 x g_(max), and once for 30 min at10,000 x g_(max). The pellet was referred to as microparticles. Thesupernatant were mixed with endogenous exosome depleted skim milk (1:1by volume) and centrifuged for 60 min at 100,000 x g_(max). The pelletwas collected, resuspended and the exosomes collected by sucrosegradient centrifugation. Exosomes were then washed with endogenousexosome depleted skim milk once and then resuspended in PBS. Purity andintegrity of sucrose gradient-isolated exosomes was analyzed using aHitachi H7000 electron microscope (Electronic Instruments, Akishima,Japan). To further minimize potential contamination during exosomalpurification, exosomal endotoxin levels were quantified using theLimulus amebocyte lysate assay (Associates of Cape Cod Inc., Falmouth,MA) according to the manufacturer’s protocol. The concentration ofexosomes was then determined using the Bio-Rad (Hercules, CA) proteinquantitation assay kit with BSA as a standard.

Labeling of Grape-derived Exosomal-like Particles (GELPs). GELPs werelabeled using an odyssey fluorescent dye IRDye800 kit (LI-CORBiosciences, Lincoln, NE) or DIR dye kit (Invitrogen, Carlsbad, CA).C57BL/6j mice were gavaged with the IRDye 800CW-labeled or DIR-labeledGELPs (100 µg), and mice were imaged using a prototype LI-COR imager(LI-COR Biosciences) for IRDye 800CW-labeled GELPs or for DIR-labeledGELPs. Mice receiving nonlabeled GELPs were used for establishing abaseline background.

FACS analysis. For cell surface marker staining, isolated cells wereblocked at 4° C. for 5 minutes with 10 µg/ml of mouse Fc block (BDBiosciences, San Jose, CA) and then reacted with variousfluorochrome-labeled antibodies including appropriate isotype controlsfor 30 minutes at 4° C. After washing twice, cells were fixed andanalyzed using a FACS Calibur flow cytometer (BD Biosciences, San Jose,CA). Data were analyzed using FlowJo software (TreeStar, Ashland, OR).The following antibodies were used for immunostaining: EPCAM, CD 14,F4/80, and annexin V (eBiosciences, San Diego, CA).

Preparation of exosomal curcumin and quantification of curcumin.Exosomal curcumin was prepared by mixing curcumin with GELPs in PBS.After incubation at 22° C. for 5 min, the exosomal curcumin wasseparated from unbound curcumin by sucrose gradient centrifugation asdescribed. To determine the concentration of curcumin in the samples,samples collected from mice were precipitated with emodin to removeproteins and analyzed using a method described previously. Proteinconcentration was calculated based on the absorbance units with respectto the concentration of curcumin in the standard curve.

ELlSAs. Supernatants, colon homogenates or serum were analyzed for thepresence of IL-6, MIP1, IL-1β (all BD Biosciences, San Jose, CA), as perthe manufacturer’s suggested protocol.

Coomassie Blue staining of protein gels. Total protein extracts fromGELP, Grapefruit-derived Exosomal-like Particles (GFELP) and TS/Aexosomes (100 µg/lane) were prepared using cold RIPA buffer (Pierce,Rockford, IL) and separated by 10% SDS PAGE. The gels were stainedsubsequently with Coomassie Blue and scanned using an Odyssey Scanner(Li-COR).

Induction of arthritis. DBA/1j female mice (Jackson Lab) were immunizedat 7 weeks of age at the base of the tail with 200 µg of bovine collagenII (CII) dissolved in 100 µl of 0.05 M acetic acid and mixed with anequal volume (100 µl) of CFA (Chondrex Inc., Redmond, Washington, USA).

Treatment protocols for mice immunized with CII. Three weeks afterchallenge with collagen II and one day after a boost with incompleteadjuvant, mice received the following oral treatments every other dayfor 20 days: (1) GELP-Cur (4 mg/kg of body weight dose was used based ona dose-response curve); (2) Free curcumin (4.0 mg/kg of body weight),(3) GELPs, and (4) PBS (n = 10 per group). Blood was collectedimmediately before and 1 h after treatment on days 1, 10 and 20, andurine and feces were collected on days 1, 10, and 20, as well as the daybefore treatment. Blood samples were collected for analysis ofglutathione-s-transferase (GST) and alanine transaminase (ALT) activityon the last day of treatment. Curcumin in plasma, urine, and feces wasquantified using reverse-phase HPLC analysis. At least ten mice wereincluded per group.

Evaluation of the development of arthritis and joint damage. Caliperswere used to determine the diameter of each paw of each mouse every dayfor 20 days. Paw swelling was determined as the increase in diametercompared with the diameter at the initiation of the experiment. Theseverity of arthritis was graded according to the following scale: 0,normal with no swelling and erythema and no increase in joint diameter;1, slight swelling and erythema with 0.1 to 0.3-mm increase in jointdiameter; 2, swelling and erythema with 0.3 to 0.6-mm increase in jointdiameter; 3, extensive swelling and erythema with 0.6 to 0.9-mm increasein joint diameter; 4, pronounced swelling and erythema with 0.9 to1.2-mm increase in joint thickness or obvious joint destructionassociated with visible joint deformity or ankylosis. Each limb wasgraded, resulting in a maximum clinical score of 16 per animal andexpressed as the mean score on a given day.

LPS mouse septic shock model. Curcumin (Cur) or GELPs-Cur (4 mg/kg ofbody weight) was injected intraperitoneally (IP) into C57BL/6j micetogether with LPS (18.5 mg/kg, Sigma-Aldrich). PBS and GELPs alone(equal to the amount in GELPs-Cur) were used as controls. Mousemortality was monitored over a period of 4 days.

DSS plus AOM induced colon cancer in a mouse model. Colitis-associatedcolorectal cancer (CAC) was induced as described below. Six to sevenweek old female mice were injected intraperitoneally (IP) with 12.5mg/kg azoxymethane (AOM; Sigma-Aldrich Chemical Co, St. Louis, MO).Beginning on the same day, mice were provided 0% dextran sulfate sodium(DSS; MP Biomedicals, Solon, OH, molecular weight 35,000-50,000 kDa)water for 7 days followed by 14 days of untreated drinking water. Micewere subjected to two more DSS water - untreated drinking water cyclesfollowed by untreated water for 28 days. Three days after the last DSScycle, the mice were randomly grouped for treatments as described below:The treatment groups were: Group 1: grape exosomes containing curcumin(GELP-Cur, 4.0 mg/Kg of body weight); Group 2: grape-fruit exosomescontaining curcumin (GFELP-Cur, 4.0 mg/Kg of body weight) and Group 3:curcumin in liposomes (4.0 mg/Kg of body weight). The mice were gavagedtreated every other day for 9 days (10 mice/group). At the terminationof the study, mice were sacrificed by CO₂ asphyxiation and all organswere carefully inspected for macroscopic pathological lesions. Theintestines, liver, and spleen were removed, and fixed in 10% bufferedformalin for a minimum of 24 h. The colon was excised, flushed withsaline, the length measured, cut open longitudinally, flattened onfilter paper and washed with saline. Macroscopic inspection of thecolons was carefully carried out, with the number, size and location ofvisible tumors recorded.

Statistical analysis. Statistical analysis of the survival curves wasperformed using a log-rank test. Statistical analyses of tumor sizeswere performed using the Mann-Whitney test. Comparisons of one-variabledata were performed using a two-tailed unpaired Student’s T-test. Whenthe F-test indicated variances differed significantly, Welch’scorrection to the Student’s T-test was employed. Comparisons of twovariable data were performed using a two-way ANOVA with Bonferroni posttest. All tests were performed using 95% confidence intervals. Data wereexpressed as the means ± SEM. ^(∗) = p<0.05, ^(∗∗) = p<0.01, ^(∗∗∗) =p<0.001.

Example 1 - Analysis of Quantities of Exosome-Like Particles in Fruit

Based on the reports that plants also release exosome-like particles(nanoparticles), edible fruits were screened and it was found thatgrapes release large quantities of nanoparticles. In brief, under GoodLaboratory Practice (GLP) guidelines, fresh grapes were purchased fromthe market, washed and sterilized under UV lamps for 30 min. The juicefrom crushed grapes was collected into centrifuge tubes. GELPs werepurified using gradient centrifugation with three bands being visible(FIG. 1A); the middle band (density = 1.12-1.23 g/ml) was washed andresuspended in PBS. The concentration of GELPs was determined byanalyzing protein concentration using the Bio-Rad protein quantitationassay kit with BSA as a standard. The sucrose-separated GELPs wereexamined with a TECNAI T-12 electron microscope (FEI Co., Hillsboro,Oregon). EM examination indicated that grapes produce particles 50-100nm in size with features characteristic of exosomes (FIG. 1B). Using aprotocol identical to the one described above, grape-fruit nanoparticles(FIG. 1C) were also isolated. Quantification of the exosomes suggestedthat both grapes (2.2 g/pound of grape), and grapefruit (1.6 g/pound ofgrapefruit) release large quantities of nanoparticles. Coomassie bluestained 10% polyacrylamide gels of fruit nanoparticle samples showedthat both of the fruit-derived nanoparticles have a differentcomposition from exosomes derived from the mouse TS/A breast tumor cellline (FIG. 1D).

Example 2 - Analysis of Lipid Content in Fruit Exosomes

Using a tandem mass spectrometer with a collision cell (chamber wherefragmentation occurs) between the two mass specs, the FELPs samples wesubmitted to the Kansas Lipidomics Research Center, (Kansas StateUniversity, Manhattan, KS) for lipid analyses. Among the lipidsdetected, phosphatidylcholines (PC) are enriched in exosomes isolatedfrom tomatoes (20.8%), and grapefruit (28.5%). Phosphatidylethanolamines(PE) are enriched in exosomes isolated from tomatoes (18.1%), andgrapefruit (26.09%), grapefruit (45.5%). Phosphatidic acid (PA) isenriched in both exosomes and microparticles isolated from tomatoes(40.5%, 46.5%), and grapefruit (53.1%, 73.4%), but significantly lowerin grapefruit exosomes and microparticles (2.4% and 1.9%).

Example 3 - Analysis of the Ability of Intestinal Epithelial Cells andMacrophages to Take Up Grape Exosomes

DBA/1j female mice were immunized at 7 weeks of age at the base of thetail with 200 µg of bovine CII dissolved in 100 µl of 0.05 M acetic acidand mixed with an equal volume (100 µl) of CFA (Chondrex Inc., Redmond,Washington, USA). Three weeks later, DBA/1j mice that had been immunizedwith collagen II for 3 weeks to induce arthritis were administeredOdyssey 800 dye-labeled GELPs orally. Control groups of DBA/1j mice thathad been similarly immunized with collagen II to induce arthritis wereadministered the same amount of free Odyssey 800 dye or PBS. Twelvehours after oral administration of the GELPs, the results of imaginganalysis showed that Odyssey 800 dye-labeled GELPs bind to the intestine(FIG. 2A). The binding was GELP specific as the binding signal generatedfrom mice given free dye or PBS was much weaker. Further FACS analysisof cells isolated from the intestines provided the evidence that most ofthe PHK67⁺ cells are also EPCAM⁺ (FIG. 2B), which is a marker forepithelial cells. It was then determined whether the intestine-boundGELPs can enter into peripheral blood as do other nanoparticles. Toidentify the types of cells that take up GELPs, the sucrose-purifiedGELPs were labeled with PKH67 as described previously. Labeled GELPs orfree dye were administrated orally to 10-week-old DBA/1j mice that hadbeen immunized with collagen II for 3 weeks to induce arthritis.Analysis of the distribution labeled GELPs indicated that 3 h afteradministration GELP-positive cells were observed in the peripheral bloodand spleen (CD14⁺PHK67⁺), but not in the lung or liver (FIG. 2C). It wasfurther observed that the higher the amount of GELP administered, ahigher percentage of PHK67⁺ cells was present in the peripheral blood.Twenty-four hours after oral administration, the mice were killed. Anaccumulation of GELP-positive cells was observed in frozen sections ofthe synovial tissue of the inflamed joints of the mice withcollagen-induced arthritis (FIG. 2D) but not naive DBA/1j mice. FACSanalysis of collagenase-digested swollen joints indicated that withinthe gated R1 region more than 65% of the PHK67⁺ cells were F4/80⁺macrophages (FIG. 2E). The percentage of F4/80⁺ macrophages wasdependent on the dose of GELPs administrated orally (FIG. 2E). Thesedata suggested that GELPs are taken up by intestinal epithelial cells,as well as CD14⁺ cells, and that these CD 14+ cells carrying GELPmigrate to areas of inflammation.

Example 4 - Analysis of Use of GELPs as Curcumin Carriers to TreatCollagen II-Induced Arthritis of DBA/1j Mice Without Toxicity

Since GELPs are selectively taken up by monocytes, it was conceivablethat the GELPs could be used as a vehicle to deliver anti-inflammatorydrugs in a monocyte-specific manner. Initially, curcumin was selected asit is an anti-inflammatory agent that is hydrophobic and could complexwith GELPs, which are lipid-enriched, hydrophobic nanoparticles.Moreover, curcumin has therapeutic effects on both collagen II-inducedarthritis and human rheumatoid arthritis. In these experiments, it wasobserved that the GELPs bound curcumin (FIG. 3A). Using an HPLC methodas described to quantify curcumin bound to GELPs, the results indicatedthat curcumin bound to GELPs in large quantities (2.5 ± 0.3 mg/g ofGELPs). To determine whether treatment of mice with GELP-complexedcurcumin (GELP-Cur) can reduce or eliminate arthritis in a mouse model,the collagen II-induced inflammatory arthritis of DBA/1j mice was usedfor proof of concept. Arthritis was induced using a standard protocol.Three weeks after challenge with collagen II and one day after a boostwith incomplete adjuvant, mice received the following oral treatmentsevery other day for 20 days: (1) GELP-Cur (4.0 mg/Kg of body weight);(2) Free curcumin (4.0 mg/kg of body weight), (3) GELPS, and (4) PBS (n= 10 per group). Blood was collected immediately before and 1 h aftertreatment on day 1, 10 and 20, and urine and feces were collected ondays 1, 10, and 20, as well as the day before treatment. Blood sampleswere collected for analysis of GST and ALT activity on the last day oftreatment. Curcumin in plasma, urine, and feces was quantified usingreverse-phase HPLC analysis as described previously.

Levels of Curcumin in Blood and Excreta. The results of HPLC analysis ofplasma indicated that mice receiving GELP-Cur had curcumin amounts of 78± 4.2 nmol/L 1 h post-oral administration on day 1 and at similar levels1 h post-administration on days 10 and 20. Curcumin was not detected inthe plasma of the groups of mice treated with free curcumin, GELPsalone, or PBS. In contrast, analysis of urine indicated that curcuminwas present at much higher levels in the group of mice receiving freecurcumin (0.7 ± 0.1 µmol/L) than those that received GELP-Cur (0.02 ±0.01 µmol/L). In day 1 fecal samples, the curcumin levels from micetreated with free curcumin were 101 ± 11.2 nmol/g dried feces; whereas,the levels in the GELP-Cur-treated group were 15 ± 1.1 nmol/g driedfeces. The liver enzyme levels for ALT (36 ± 8.1 U/I) and GST (25 ± 5.2)were not significantly different among the treatment groups: GELP-Cur (p= 0.56), curcumin (p = 0.46), and GELPs (p = 0.78) in comparison withthe group treated with PBS.

Biological affects of oral curcumin. The sera levels of TNF-α (FIG. 3B)and IL-6 (FIG. 3C) were much lower in the group of mice treated withGELP-Cur than in the other treatment groups. This was supported by lowerlevels of both TNF-α (FIG. 3D) and IL-6 (FIG. 3E) in supernatants whenwhole blood samples were cultured for 24 h with LPS (100 ng/ml). The invivo inhibition of induction of IL-6 (FIG. 3F) or TNF-α correlated withthe induction of apoptosis in monocytes that were CD 14⁺PI⁺AnnexinV⁺(FIG. 3F). In addition to the reduction in IL-6 and TNF-α and theinduction of monocyte apoptosis after GELP-Cur treatment, there was lessmacroscopic evidence of arthritis in the GELP-Cur-treated mice whencompared with mice treated with free curcumin, GELPs only, or PBS.

Collectively, the data generated in this study supports the notion thatcurcumin strongly binds to GELPs leading to an enhancement of curcuminstability and bioavailability. After GELP-Cur treatment, there was lessmacroscopic evidence of arthritis in the GELP-Cur-treated mice whencompared with mice treated with free curcumin, or PBS.

Example 5 - Analysis of Use of GELPs as Curcumin Carriers to TreatSeptic Shock

GELPs can be used as curcumin carriers to protect mice against LPSinduced septic shock. Using an identical treatment protocol as describedabove, GELP-Cur-treatment led to 100% protection of C57BL/6 mice (10mice/group) against LPS induced sepsis in a murine model. In contrast,oral administration of free curcumin at an amount equal to that inGELP-Cur had no effect on morality (8/10 mice died) as compared to micetreated with PBS (9/10 mice died).

Example 6 - Analysis of Ability of GELP-Cur Treatment to DecreaseDevelopment of Colitis-Associated Colon Cancer

To examine the therapeutic effects of GELP-Cur during colitis-associatedtumorigenesis, a well-established model of colitis-associated cancer(CAC) was used. Mice received a single injection of azoxymethane (AOM,10 mg/kg body weight), followed by administration of three cycles ofdextran sulfate sodium (DSS, 2% in water) as outlined in FIG. 4 .GELP-Cur or GFELP-Cur (gavaged at 4 mg/kg of body weight, every otherday for 3 weeks) treated mice had a significantly lower tumor burdenthan mice treated with free curcumin (FIG. 5A). The average tumor numberper mouse was more than two-fold decreased in GELP-Cur or GFELP-Curtreated mice compared to curcumin treated mice (3.1 vs. 8.5, p<0.01). Inaddition to the decrease in tumor burden, GELP-Cur or GFELP-Cur treatedmice also had a significantly lower number of larger tumors (>1 mm²)(p<0.05). Significantly decreased serum levels of IL-6 and MIP-1 werealso detected in whole colon homogenates of GELP-Cur or GFELP-Curtreated mice compared to curcumin treated mice (p<0.05) (FIGS. 5B and5C).

Discussion of Examples 1-6

Oral delivery using artificially-synthesized nanoparticles has beenidentified as an alternative delivery system that can overcome the poorabsorption of curcumin through the intestines; however the challengesrelated to efficient nanoparticle delivery, clearance and toxicity mustbe overcome before nanoparticles can be used in a clinical setting. Inthis regard, it is appreciated that exosomes are released from manytypes of cells and theoretically could be used as a biodegradabledelivery vehicle. Data suggest that the stability, solubility andbioavailability of curcumin is enhanced dramatically by encapsulatingthe curcumin in mammalian-derived exosomes (Exo-cur). It has furtherbeen demonstrated that mice treated orally with Exo-cur are completelyprotected against LPS-induced septic shock. Although promising, thefeasibility of that approach for treatment of patients is limited by theneed for large scale production of mammalian exosomes. In the foregoingstudies, alternative sources of exosomes were thus explored and it wassurprisingly found that several edible fruits are a source of largequantities of nanoparticles (also referred to as exosome-likeparticles). This indicated the possibility that fruit-derivednanoparticles might be a potential delivery vehicle for use in clinicsettings. It was found that nanoparticles released from the flesh ofgrapes and other fruit as well could be of particular usefulness as atherapeutic delivery vehicle based on data presented in the foregoingExamples, indicating that: (i) oral administration of grapenanoparticles at doses of up to 40 mg/kg of mouse body weight was safewithout evidence of liver damage; (ii) using mammalian T cell-derivedexosomes, grape-derived exosomes could be used to encapsulate curcuminand oral administration of those grape exosome-like particles (GELP)carrying curcumin (GELP-Cur) protected mice against LPS-induced septicshock and did so by specifically targeting inflammatory myeloid cells;(iii) oral administration of GELP-Cur protected mice against collagenII- induced arthritis, and (iv) oral administration of GELP-Cur had asignificant therapeutic effect on AOM/DSS induced colon cancer. The dataalso suggested that grape nanoparticles were preferentially taken up byintestinal epithelial cells, monocytes, and macrophages. As such, it wasfurther believed that the foregoing strategy could be applied to anydisease in which activation of macrophages and monocytes played a rolein the disease process.

In addition, the foregoing studies indicated that the exosomalcompositions could play a role in the development of a non-invasivefood-derived system for the delivery of curcumin and perhaps otherdrugs, as well as multiple drug carriers. The foregoing results providednovel approaches to eliminate/reduce chronic inflammation ininflammatory tissue and yielded a new, cost-effective, and practicaltherapeutic method for treatment of inflammatory-related diseasesincluding inflammatory RA in patients; thus improving the health statusof that patient population. The studies also had more generalimplications in terms of the basic biology of the effects of diet andthe consumption of edible fruits and vegetables on health and disease.Exosome-like nanoparticles are released from many types of fruits orplants and it is conceivable that these exosomes may play key roles inpromoting the delivery of specific nutrients, phytochemicals, and otheringested bioactive compounds (beneficial or otherwise) to the immunesystem.

Example 7 - Analysis of Ability of GELP Treatment to Protect MiceAgainst DSS-Induced Mortality and Colitis

Treatment: To test whether fruit derived nanoparticles preventedDSS-induced colitis, C57BL/6 were given 3% DSS (MW 36,000-50,000; MPBiomedicals, Solon, OH) dissolved in reverse osmosis water ad libitumfor 7 d (i.e., one cycle of DSS). Gavages of PBS, GFELP or GELPtreatments (100 ug /mouse/gavage in 200 ul PBS) were given for fiveconsecutive days starting at day 0 after the administration of 3% DSS.On day 8, mice were returned to untreated drinking water and sacrificedon day 15 since all PBS-fed mice were dead within day 15 after drinking3% DSS contained water.

Evaluation for treatment effects: Mice were checked for rectalprolapse/macroscopic bleeding, and accumulated mortality. The presenceof blood in the stools was assessed by a guaiac paper test (ColoScreenOccult Blood Test, Helena Laboratories, Beaumont, TX) using a 0 to 4scale: 0 = negative, 1 = faintly blue, 2 = moderately blue, 3 = darkblue, 4 = fecal blood visible to the eye. Each mouse was scored 4 timesper day, resulting in a maximum clinical score of 16 per animal andexpressed as the mean score on a given day.

After mice were sacrificed, cryostat sections (5 µm) of the intestinaltissue were prepared and stained with hematoxylin and eosin. The stainedsection was analyzed in a blind fashion with a light microscope (BX10,Olympus). The pathophysiology of the tissue was characterized by cryptloss and thickening of the villus length from the base of the villus tothe villus apex of GELP and PBS gavaged intestines was measuredmicroscopically. The intestinal tissues were also fixed and stained forcell proliferation by BRDU staining, immunohistologically stained forthe stem cell marker, BMI1, and β-catenin using a method as described.

Permeability test: At day 7 post-DSS treatment, mice were sacrificed viaCO₂ asphyxiation and intestinal tissues were resected. Uponequilibration of the tissue and measurement of electrical parameters,4-kD FITC-dextran was added to the mucosal side to achieve a finalconcentration of 0.01 mM. Permeability (flux) was determined afterremoval of medium from the serosal compartment at the end of a 90 minuteexperimental period. The medium was then assessed for fluorescence usinga microplate fluorescence reader (FL-500, BIO-TEK, Vermont, USA) at anexcitation wavelength of 485 nm and an emission wavelength of 530 nm.

Immunoblotting. Isolated jejunal IECs and jejunal tissues were saved.Total cellular protein extracts from intestinal tissues, CT26 coloncells (ATCC) or MCA38 colon cells were prepared using cold RIPA buffer(Pierce, Rockford, IL). Expression of total and phosphorylated GSK3βwere detected using a method as described previously.

Immunohistochemistry. Frozen tissue sections from mouse jejunum (4 µm),CT26 (ATCC) or MCA38 (ATCC) colon cells were prefixed inparaformaldehyde. Tissue sections were stained with anti- β -catenin,BMI1, and anti-BRDU antibodies. 4′,6-diamidino-2-phenylindoledihydrochloride (DAPI) was used for nuclear counterstaining. Images werecaptured using a Zeiss microscope and Axioviewer image analysis software(Carl Zeiss Corp).

Bacterial counts in the liver. For the determination of bacterial burdenin mouse liver, at day 7 after 3% DSS treatment, 5 mice from eachtreated group were sacrificed, and livers were homogenized. Dilutions ofthe homogenates were plated on cystine heart agar plates and incubatedfor 24 h at 37° C. Bacterial colonies were counted and recorded as CFUper ml per gram of tissue.

Statistical Analysis. Results were presented as the mean ± SEM. Datawere analyzed using analysis of variance, 2-tailed Student’s t test, andthe Mann-Whitney test as appropriate (Prism, GraphPad, San Diego, CA). Pvalues of ≤0.05 were considered significant.

GELP gavaged mice and PBS mice were provided a 3.0% solution of DSS inthe drinking water for 7 d and observed for 15 days. All DSS-treatedmice developed colitis, as indicated by being severely depressed (FIG.6A) and based on stool quality (bloody stool, FIG. 6B). At day 13, morethan 80% of the mice gavaged with PBS were dead. In contrast, GELPgavaged mice showed no signs of depression and less blood was detectedin stool samples (FIGS. 6A and 6B). There were no GELP treated mice deadby day 15 (FIG. 6C). Mice were sampled and killed at day 7 after beingprovided 3% DSS in water and histological and intestine permeabilityanalyses were performed. As expected, colon length in PBS-gavagedcontrol mice was significantly shorter (FIG. 6D, P < 0.01) compared withnon-DSS untreated control mice. However, in GELP gavaged mice, colonlength was the same as non-DSS treated control mice (FIG. 6D).Histological scoring of colonic sections showed less severe damage ofintestinal tissue in GELP gavaged mice compared with PBS gavaged mice(FIG. 6E) with a significantly thicker mucous layer in the GELP gavagedmice (FIG. 6E).

Intestinal epithelial cells, in addition to promoting digestion andabsorption of nutrients, perform essential barrier functions by tightlyregulating intestinal permeability. It was found that there wasremarkably elevated permeability in the small intestine after 7 daysconsumption of 3% DSS in drinking water and the increase wassignificantly higher in PBS gavaged mice compared to GELPs gavaged mice(FIG. 6F). Consistently, a significantly higher amount of bacteriatranslocated to mesenteric lymph nodes (MLN) in PBS gavaged micerelative to GELP treated mice after 7 days consumption of 3% DSS water(FIG. 6H), demonstrating that GELPs treatment prevented DSS inducedintestinal barrier dysfunction after a DSS challenge. DSS inducedcolitis is accompanied by an increase in the levels of inflammatorycytokines, including IL-6 and IL-1β. Levels of circulating cytokines inthe serum were measured, and it was found that IL-6, as well as IL-1βwere significantly higher in the peripheral circulation in PBS gavagedmice (FIG. 6G) when compared to GELP gavaged mice. This indicated thatGELP treatment prevented the induction of inflammatory cytokines thatplay an essential role in DSS induced colitis.

Next, it was determined whether GELP treatment effected the localizationof β -catenin in intestine epithelia. It is appreciated that activationof the Wnt- β -catenin pathway promotes the regeneration of epithelialcells after DSS induced intestinal injury, including stem cellregeneration. In this regard, it was hypothesized that GELP acceleratedthat regeneration process by enhancing β -catenin mediated activation ofthe Wnt- β -catenin pathway. One of the initiating events of activatingthe Wnt- β -catenin pathway is nuclear translocation of β -catenin, so,to determine whether GELP treatment had effects on the translocalizationof β -catenin in intestinal epithelium, the localization of intestinalβ-catenin was analyzed in PBS- versus GELP-treated mice. Immunostainingof sections from these mice 7 days after being provided 3% DSS indrinking water showed β-catenin stains much stronger in the cytosol andnucleus of mice treated with GELP than in mice that received PBS (FIGS.7A and 7B). Induction of phosphorylation of GSK3β is required fortranslocation of β-catenin from the cell membrane to the nucleus. Next,it was tested whether GSK3β is phosphorylated after mice were treatedwith GELPs. The results of western blot analysis showed that thephosphorylated form of GSK3β is induced in the small intestine of micetreated with GELPs (FIG. 7C). These in vivo results were furtherconfirmed by immunohistological staining (FIGS. 7D and 7E) and westernblot analysis (FIGS. 7F and 7G) of β-catenin in mouse colon cell lines,including CT26 (FIG. 7D) and MCA38 (FIG. 7E). The results indicated thatβ-catenin is translocated from the cell membrane into the cytosol andnucleus and that the phosphorylated form of GSK3β was induced when thecells were exposed to GELPs but not PBS. Collectively, these resultsfurther indicated that GELP treatment led to the inactivation of GSK3βand caused β -catenin nuclear translocation in both in vitro as well asin vivo mouse models. This was believed to be important as β -catenintranslocation is required for activation of the Wnt signaling pathwaythat plays an important role in generation of intestinal stem cells(ISCs), and ISCs are important for maintaining tissue homeostasis andreplacing lost cells in response to tissue damage such as DSS inducedepithelial cell damage

Next, it was tested whether epithelial renewal driven by stem cellsplayed a role in GELP- mediated protection of mice against DSS inducedcolitis. A polycomb transcriptional repressor family member (BMI1) hasrecently been identified as a specific marker of stem cells inepithelium. To investigate whether BMI1 expression is altered in thesetting of colitis, C57BL/6 (WT) mice administered 3.0% DSS in thedrinking water were gavaged with GELPS or PBS (as a control) every otherday for 6 days. The mice were examined for levels and cellularlocalization of BMI1 and cell proliferation at day 7 after thetreatments. In contrast to the expected BMI1 immunoreactivity, there wasa generally very low frequency of BMI1-positive cells, which wereusually found in the base region of the intestinal crypts (FIG. 8A,left), with no expression at the luminal surface. In contrast, there wasa very obvious significant increase in the frequency of stronglyBMI1-positive cells throughout the crypts of grape exosome gavaged mice(FIG. 8A, right). Increased levels of BMI1 correlated with an increasein BRDU incorporation in the same regions of the intestinal crypts (FIG.8B) and an increase in cellularity in the base region of the crypts ofHE stained intestinal tissue sections (FIG. 8C). These results indicatedthat GELP treatment led to the promotion of intestinal epithelial cellproliferation and the cells were likely to be BMI1 positive stem cells.

Example 8 - Use of Fruit-Derived Exosomes as a Noninvasive DetectionMethod for Colon Cancer-Associated Inflammation.

Colorectal cancer (CRC) is the second leading cause of cancer mortalityin the United States, and the third most common type of cancer in menand women. Diagnosis of CRC indicating lymph node metastasis, isassociated with poor survival rates, demonstrating the necessity forearly detection. Currently, the most commonly used screening techniquesare fecal occult blood testing (FOBT), sigmoidoscopy, colonoscopy, andcomputed tomographic (CT) colonography, all of which possess bothadvantages and disadvantages. All of these methods either fail to detectmost early precancerous polyps and cancerous lesions or are veryinvasive. In addition, these methods are both time- and cost-intensiveand require substantial expertise. Based on the results described below,it was found that using DIR dye labeled GELP is useful foridentification of CRC in the early stages of disease or even beforedisease onset, thus allowing for both the elimination from expensivetherapeutic studies, as well as the assessment of therapeutics atvarious stages of disease.

Briefly, in these studies, mice received a single injection ofazoxymethane (AOM, 10 mg/kg body weight) or PBS (as a control), followedby administration of three cycles of dextran sulfate sodium (DSS, 2% inwater) as outlined in FIG. 4 . Mice (8 mice/group) were imaged at week1.0, 3.0, 5.0, 9.0 and 13.0 weeks after the AOM administration using aCarestream Molecular Imaging System (Carestream Health, Inc). Mice wereanesthetized with 2% isoflurane in oxygen through a nose cone during theimaging. Body temperature was supported with warm air circulating in themagnet bore (SA Instruments, Inc, Stony Brook, NY). Immediately afterthe imaging, mice were killed and carefully dissected without disturbingthe position of abdominal contents in situ, and then examined formacroscopic tumor formation. The results showed that macroscopicallyvisible tumor was not evident until week 11. In contrast, starting atweek 5 after AOM administration, mice gavaged with DIR-dye labeled GELPhave a much stronger signal in the colon than of PBS control mice (FIGS.9A and 9B).

Example 9 - Clinical Investigation of GELP-Cur Treatment for RheumatoidArthritis

To investigate the safety of GELP-Cur treatment for rheumatoid arthritis(RA) patients and to generate data in support of conducting a largeclinical trial. An initial clinical trial is performed. Eligiblepatients must meet the most recent American College of Rheumatology(ACR) criteria for rheumatoid arthritis, be at least 18 years of age,and have had clinical features of RA for less than 12 months. To beincluded, patients will have had an inadequate response todisease-modifying antirheumatic drugs (DMARDs) including anti- TNF-αtherapy, after at least three months of such treatment. Patients musthave 5 or more swollen joints and/or tender joints at the time ofenrollment. Changes in the doses of background DMARDs will not bepermitted except to avoid adverse effects. Forty-five patients areenrolled for a pharmacokinetics and safety study using GELP-Curtreatment with each dose as described in Table 1 below.

Table 1 Dosing Guidelines 1. If no one in a cohort of 15 patients (pts)treated with 5 mg/kg of body weight experiences side effects (SE), thedose will be doubled for the next cohort. 2. If 1-2 of a cohort of 15pts experiences SE, the dose will remain the same for the next cohort asthe first cohort. 3. If 3 or more of 15 pts experience SE, the dose willbe reduced to 2.5 mg/kg of body weight for the next cohort. 4. If 4 ormore pts out of 30 consecutive pts (2 cohorts of 15 pts each, all at thesame dose) experience SE, the dose will be reduced to 2.5 mg/kg of bodyweight for the next cohort.

Patients who have allergies to curcumin or grapes, and patients takingdrugs that are known to interact with grape juice are excluded. Patientsare educated on food sources of curcumin and asked to avoid all foodscontaining high concentrations of curcumin within the 14 d beforeGELP-Cur administration. Subjects complete a food checklist to verifythat they are not consuming any curcumin-rich foods before dosing.

The curcumin for the study is purchased from Sabinsa Corp. (Piscataway,NJ), which has also furnished the material for several other clinicaltrials, including a recent pancreatic cancer trial conducted at M. D.Anderson Hospital, Houston, TX. The GELP-Cur is prepared under GLPprotocol by qualified personnel. The GELP is prepared using gradientcentrifugation as described previously. The GELP is resuspended insterile 0.9% NaCl and the concentration of GELPs is determined byanalyzing protein concentration using the BioRad protein quantitationassay kit with BSA serving as the standard. Quality control proceduresinclude the precautions described previously, and analysis of potentialcontamination of the exosomes during the purification process byassessing endotoxin levels using the Limulus amebocyte lysate assay(Associates of Cape Cod, Inc.). In addition, the composition of the GELPis analyzed by HPLC to monitor the presence of flavinoids and otherpotentially bioactive molecules in the GELPs. Two-dimensional gelelectrophoresis is used to generate a protein signature for qualitycontrol purposes.

The GELP-Cur is then prepared by mixing curcumin with a standardizedamount of GELPs in 0.9% NaCl. After incubation at 22° C. for 5 min, theGELP-Cur is separated from unbound curcumin by centrifugation andlyophilized. The concentration of curcumin in the GELP-Cur is determinedby HPLC analysis and the biological activity of the GELP-Cur analyzed bymeasuring inhibition of TNF-α and IL-6 production after LPS stimulationof a macrophage cell line (RAW264.7). Aliquots are stored at -20° C. andadministered to patients immediately after suspension in drinking water.

Curcumin used for patients is seldom administered in a pure chemicalform. Rather, it typically consists of three separate curcuminoidscomposed of curcumin itself, as well as demethoxycurcumin andbisdesmethoxycurcumin. To determine the qualitative and quantitativepresence of these curcuminoids in the GELP-Cur product as prepared anddescribed above and to be used for this clinical trial, the GELP-Curmaterial is separated using a Gemini 5 um C18 (2 × 100 mm) analyticcolumn (Phenomenex) and a linear acetonitrile/methanol/0.2% formic acidgradient. The amount of curcuminoid is detected using a Waters QuattroUltima tandem mass spectrometer equipped with electrospray-positiveionization capability in the mass spectrometry facility of University ofLouisville. All three compounds are quantified using standardcalibration curves prepared from reference standard materials obtainedfrom Sigma-Aldrich. Calibration curves are prepared by making a 1 mg/mLstock solution of the authentic materials in methanol and then seriallydiluting the stock solutions to 1,000, 500, 100, 50, 10, 5, and 1 ng/mLin 50:50 methanol/0.2% formic acid. Calibration curves are then preparedusing the mass spectrometry quantification software program.

For the GELP-Cur treatment of patients, the study design is based on aconventional “15 + 3” dose-escalation model, with patients receivingoral GELP-Cur daily, at a dose of 5 mg/kg of body weight, beginning onthe day of enrollment until day 180. Each subsequent cohort will receivea dose of GELP-Cur determined by the guidelines in Table 1. A maximumdose of 20 mg/kg of body weight will be pre-selected during the designof the study because this is within the safety dose of curcumin that hasbeen used in clinical trials. Study medication is administered orallydaily for up to 6 months. Subjects take the dose with 8 fl. oz. ofwater.

All clinical assessments are performed at the University of LouisvilleHospital. Blood and urine samples are collected at 0 (immediatelypreceding GELP-Cur administration), 6, 12, and 24 hours post-dose andbefore a second dose. The procedure is repeatedly applied to a secondand third escalated dose. Serum and urine concentrations of curcumin aredetermined using methods described previously. Steady-statepharmacokinetic parameters are determined, i.e., maximum observed plasmaconcentration (Cmax), minimum observed plasma concentration (Cmin),average observed plasma concentration (Cav), area under the plasmaconcentration-time curve from 0 to 24 hours (AUC₀₋₂₄), and time to Cmax(tmax). These primary model variables are estimated using SAS PROCNLINMIXED (SAS Institute), from which estimates of area under the curve(AUC), Tmax, Cmax, and t_(½) will be derived.

Safety is assessed by occurrence of adverse events (AEs) as listed inTable 2 below and by monitoring of biochemical, hematological andurinalysis parameters every month during the study period, with toxicitygraded according to the Common Toxicity Criteria version 3.0. Adverseevents, such as gastrointestinal complaints, vomiting, anorexia,headache, dizziness, insomnia, fever, and mouth ulcers are included. Forassessing the safety of the treatments, patients are monitored monthlyfor AEs, serious AEs, and clinically significant changes in vital signsand laboratory tests at each monthly visit during the study duration.Both the severity of AEs and their relation to the study treatment arenoted by the investigator. In addition, standard parameters areevaluated in serum, urine, and whole blood of all patients. In additionto standard safety parameters, samples are collected for phenotypingleukocyte subsets by FACS. Phenotyping includes neutrophils,macrophages, and T cells. The inflammatory cytokines IL-6, IL-8, IL-10and TNF-α are analyzed using a standard ELISA. Patients also aremonitored for tolerability and efficacy at monthly intervals from thestart of dosing through the completion of the study, as well as at afollow-up visit 6 months after completion of the study.

Table 2 List of Adverse Events • General: weight loss, fatigue, sweating• Gastrointestinal: nausea, vomiting, abdominal pain, diarrhea • Skin:rash, pruritis, alopecia, oedema, flush, haematoma, erythema, dermatitis• Neuropsychiatric: psychiatric, sleep disturbance, headache, dizziness,ENT/opthalmological • Others: stomatitis/mouth ulcers, altered taste,blurred vision • Infections: urinary tract infection, respiratoryinfection, other infection with fever

There are two end points determined at six months: the proportion of RApatients with an ACR 20 response; and the proportion of patients with animprovement of at least 0.3 from baseline in the Health AssessmentQuestionnaire (HAQ) disability index (exceeding the minimal clinicallyimportant change of 0.22). An ACR 20 response indicates a decrease of atleast 20 percent in the number of both tender and swollen joints, aswell as a 20 percent improvement in at least three of the following: thepatient’s global assessment of disease activity; the patient’sassessment of pain; physical function, as assessed by the HAQ disabilityindex; the physician’s global assessment of disease activity; and theC-reactive protein level. Secondary objectives include a 50 percent and70 percent improvement in the ACR response at six months. Changes indisease activity are assessed with the use of the Disease Activity Score28 (DAS28). Clinical remission is defined by a DAS28 of less than 2.6,and a low level of disease activity will be defined by a DAS28 of 3.2 orless. The mean improvement in physical function at six months is basedon the change from baseline in the HAQ disability index. Changes frombaseline in the health-related quality of life will be assessed by theMedical Outcomes Study including physical function, pain, general andmental health, vitality, social function, and physical and emotionalhealth.

Various parameters of serum biochemistry, hematology and urine analysisare carried out on each evaluation day. Serum samples are collected onall evaluation days for quantification of pro-inflammatory modulators.If feasible, knee joint synovial fluid is collected aseptically atbaseline and at day 180 for evaluation of MMPs and inflammatory cytokineconcentrations.

All AEs are reported in a tabular format with the following headings: AEdescription, AE onset and stop date, grade, relationship to study drug,action, action comments, and outcome. This format enables a thoroughreview of events and possible causality to ensure the safety of patientsenrolled in the trial.

To investigate a potential correlation between blood and jointconcentrations of curcumin and anti-inflammation activity, plasma andswollen joint fluid samples are collected over the course of the studyfor pharmacokinetic profiling of curcumin. In period 1, blood andsynovial fluid curcumin pharmacokinetic samples are taken on days 1 and7. In period 2, blood and synovial fluid curcumin pharmacokineticsamples are collected on day 14, and additional pharmacokinetic samplesfor curcumin determination are obtained on days 28, 90 and 180.

Standard safety parameters including blood count, serum chemistry panel,and urinalysis will be determined. Concentrations of curcumin in plasmaand urine samples are collected monthly and detected using ahigh-performance liquid chromatography (HPLC)-based method with UVdetection as described previously. Assay precision and accuracy aredetermined via analysis of a standard curcumin control with more than97% purity. Concentrations of curcumin in the samples are determined bycomparison with a standard curve of curcumin.

Various parameters of serum biochemistry, hematology, urine analysis,and bone erosion by radiography are carried out on each evaluation day.Serum samples are collected monthly for quantification ofpro-inflammatory modulators and rheumatoid factor. Knee joint synovialfluid is collected aseptically at baseline and on days 28, 90, and 180for evaluation of MMPs and inflammatory cytokine concentrations. Samplesare collected for phenotyping leukocyte subsets by FACS, includingneutrophils, macrophages, and T cells. The inflammatory cytokines, i.e.,IL-6, IL- 8, IL-10 and TNF-α, are also analyzed using a standard ELISAas described previously.

The trial is designed to have more than 80% power to detect a situationin which GELP-Cur treatment yields an improvement of at least a meanchange of +0.9 standard deviation in comparison with before treatment.Under this conservative assumption, differences between groups in meanimprovement are tested using ANOVA (0 = 0.05, two-sided). With 45patients, a 95% confidence of observing at least one example of any sideeffect occurring in 10% or more of the patient population with aspecific treatment is expected.

For the primary analyses of the AEs, ACR 20 and HAQ responses, theproportion of patients who have a response are summarized according tothe treatment group. A two-sided Cochran-Mantel-Haenszel chi-square testis used to compare response rates among treated groups at the 0.05 levelof significance. For the analyses of AEs, ACR 20 and HAQ responses, allpatients who discontinue treatment are subsequently considered not tohave a response. The primary and the multiple secondary end points aretested in a pre-specified sequence after the use of a closed testingprocedure, thus controlling overall for type I error rate at the 0.05level. All reported P values are two-sided. Mean changes from baselinein the DAS28, HAQ disability index, and the scores for the physical- andmental-component summary scores of the SF-36 are compared betweentreatment groups with the use of an analysis of covariance afteradjusting for the baseline assessments. Safety is evaluated according tothe frequency of AEs, changes in laboratory values, and abnormalclinical findings. P values for safety comparisons are obtained with theuse of a chi-square test or, where appropriate, Fisher’s exact test.

Based on initial data and current literature, toxicity or severeside-effects are not anticipated. If side effects are observed in RApatients treated with GELP-Cur at the proposed lowest dose (5 mg/kg), alower dose than 5 mg/kg of GELP-Cur is used. Lower doses should stillhave therapeutic effects since 1 µM of curcumin encapsulated in GELPinduced apoptosis in more than 50% of human monocytes (FIG. 10 ). Also,the concentration of curcumin in RA patients treated with GELP-Cur isexpected to remain high during the 24 hour period after treatment. Ifthis is the case, RA patients may not need daily treatments withGELP-Cur. Based on the data expected to be generated from thepharmacokinetics study of GELP-Cur treatment, the frequency of takingGELP-Cur may be reduced which would further minimize any potentialside-effects. Additionally, a reverse correlation between jointconcentration of curcumin and disease activity is expected, and it isfurther anticipated that the data indicates whether a particular subsetof RA patients is more (or less) responsive to treatment with GELP-Curthan other subsets of patients. However, as curcumin affects manypro-inflammatory pathways, it is anticipated that a large group of thepatients are responsive. For example, RA patient’s refractory toanti-TNF-α therapy may have a similar response to GELP-Cur asnon-refractory RA patients. Since macrophages are one of the majorsources of TNF-α and other inflammatory cytokines, RA patients who areresistant to TNF-α treatment may develop a TNF-α independent pathway(s)that contributes to the progression of inflammation in RA. Using aGELP-Cur strategy will eliminate these macrophages, regardless of whichinflammatory cytokines are released.

Based on the data generated from the mouse model, it is thought that thetherapeutic effect is due to elimination of activated monocytes andmacrophages. Other types of immune cells also play a role in thepathogeneses of RA; however, the fact that monocytes preferentiallymigrate into inflamed joints in RA patients allowing for the release ofcurcumin and/or its metabolic products locally in the joints where theinfiltrating autoreactive T cells, B cells and neutrophils accumulateare utilized. Theoretically, this would suppress the activities of theseautoreactive cells. This suppression is increased as treatment continuesdue to the accumulation of curcumin and/or its metabolic products in thejoints. Initial data support the hypothesis that lower doses of GELP-Cur(200 nM) do not cause apoptosis of CD14+ cells isolated from peripheralblood of healthy subjects. FACS assay of Annex V- and PI-stained cellsshowed no effect on the viability of cells treated with 200 nM ofGELP-Cur (p = 0.002) (FIG. 10A). Furthermore, co-culture of these cellspretreated with GELP-Cur (200 nM) with T cells (5:1) suppressed T cellproliferation (FIG. 10B). In RA patients who are administered GELP-Curorally, non-apoptotic monocytes (due to carrying less GELP-Cur) migrateto inflamed joints, where curcumin is released and T cell, B cell andneutrophil activities are subsequently suppressed as illustrated in FIG.11 . If this hypothesis is true, it should be observed that theconcentration of curcumin is increased as the GELP-Cur treatment isprolonged. This hypothesis is tested as follows: (a) FACS analysis ofthe number of CD14+ and CD6S+ cells with intracellular staining of TNF-αor IL-6 in synovial fluid of RA patients receiving GELP-Cur willsignificantly be reduced as the GELPs-Cur treatment is prolonged. Thesedata are correlated with a reduction of disease progression based onACR20, ACR50 and ACR70 scores; (b) Curcumin and curcumin metabolicproducts in the synovial fluid are increased as the treatment isprolonged; (c) it is anticipated that the percentage of RA patients whoresponse to GELP-Cur treatment increase since this strategy not onlyapplies to macrophage dependent RA patients but also to T cell, B celland neutrophil dependent RA patients; and (d) using FACS analysis ofCD4/CD69/IL-2/Ki-67/IFN-γ, CD19/Ki-67/BAFF-R/TACI, CD11b/TLRs, thenumbers of activated T cells, B cells and neutrophils in the synovialfluid are expected to decrease in the RA patients receiving GELP-Cur.

Without wishing to be bound by any particular theory or belief, it isthought that the proposed studies indicate a viable alternative existsfor the development of a non-invasive fruit-derived system for thedelivery of curcumin and other drugs, as well as multiple drug carriers.The results provide a novel means to eliminate/reduce chronicinflammation in inflammatory tissue and would yield a new,cost-effective, and practical therapeutic method for treatment ofinflammatory-related diseases including inflammatory RA patients; thusimproving the health status of this patient population. Systemicinhibition of inflammation using drugs such as etanercept (Enbrel) (asoluble TNF receptor), infliximab (Remicade), and adalimumab (Humira)(anti-TNF antibodies) can result in severe adverse side effects and arecostly. Hence, orally active blockers of inflammation that are safe,efficacious, and inexpensive are urgently needed.

One of purposes of the study is to examine the efficiency of usingcurcumin as an alternative to pharmacological management of chronicmusculoskeletal pain. The proposed study evaluate the safety andtoxicity of grape exosomes carrying curcumin (GELP-Cur) as a treatmentin patients with active RA, and further identifies which subsetpopulation of RA patients is more response to treatment with GELP-Cur.The outcomes from this study will further support the rational for apotentially larger clinical trial to treat both OA and RA patients. Thesuccess of the novel approach also sheds light on using fruit-derivedexosomes for carrying other complementary and alternative agents, suchas resveratrol (similar poor solubility as curcumin), to targetinflammatory cells.

Moreover, the proposed study leads to a larger clinical study for thefollowing reasons: (1) the study as proposed leads to the identificationof a subset of RA patients who do not respond, or only partially respondto DMARD treatment, and who can be effectively and safely treated withGELP-Cur. The results generated through the study also are used as abasis for selecting a larger population of RA patients who are resistantto current treatments, and also permit longer safety trials. Although anumber of immunomodulatory agents other than anti-TNF-α therapy haveshown efficacy in patients with RA, the other agents are associated witha dose-dependent risk of gastrointestinal, cardiovascular, hematologic,hepatic, and renal adverse events.

Example 10 - Analysis of Fruit-Derived Exosomal-Like Particles forNucleic Acid Delivery

Artificial systems including liposomes have been developed for thedelivery of transgenes to mammalian cells in vitro and in vivo. However,all of the methods developed to date have too low of a transfectionefficacy in vivo and in vitro or they are cost prohibitive for genetherapy use in clinical practice. Based on the results described below,it has been found that using grapefruit-derived liposomes is effectivefor delivery of nucleic acids in both in vitro cell-culture systems andin vivo. The in vivo delivery is successful when done repeatedly even inthe presence of sera. This technology eliminates the need for expensivetherapeutic studies as well as the assessment of high transfectionefficiency without causing cytotoxicity. More specifically, the in vitrostudies show that grapefruit-derived liposomes composed of optimizedratios of lipids (phosphatidylethanolamine, phosphatidylcholine,phosphatidylserine, monogalactosyldiacylglycerol (MGDG), anddigalactosyldiacylglycerol) result in higher transfection efficacy thanany of the other popular transfection agents on the market, includingLIPOFECTAMINE® 2000 (Invitrogen) and FUGENE® HD (Roche). Furthermore,unlike other transfection agents, grapefruit-derived liposomes do notcause any detectable cytotoxicity and the presence of sera does notinterfere with transfection efficiency. Also important is the fact thatthe methodology is much less expensive (less than $0.01/per reactionversus $3.50/reaction for current commercial products, calculated basedon the cost for the transfection of one 6-well plate). Therefore, themethodology and resulting product could be used much more efficientlyand economically for the in vitro and in vivo delivery of siRNA, miRNAs,and mammalian expression vectors for gene therapy without causingnon-specific cytotoxicity.

In these studies, grapefruit exosomal and microparticle lipids werefirst extracted. In brief, 10 mg of grapefruit exosomes ormicroparticles were suspended in 3 ml of preheated (75° C.) isopropanolcontaining 0.01% BHT (butylated hydroxytoluene, e.g., Sigma). After 15min of incubation, 1.5 ml of chloroform and 0.6 ml water were added tothe reaction, the mixture vortexed; and then agitated (shakingincubator) at 22° C. for 1 hour at which time the lipid extracts weretransferred with glass Pasteur pipettes to glass tubes with Teflon-linedscrew-caps. Four ml of chloroform/methanol (2:1) containing 0.01% BHTwas then added to each tube and the mixture shaken for 30 min. Thisextraction procedure was repeated 5 times. One ml of 1 M KCl was addedto the combined extracts, the mixture vortexed or shaken, centrifuged,and the upper phase discarded. Two ml of water was then added, themixture vortexed or shaken, centrifuged, and the upper phase discarded.Tubes containing the lipid extract were then filled with nitrogen gas,stored in a freezer until transported on dry ice to the KLRC AnalyticalLaboratory for MS/MS identification of lipids.

Upon obtaining the results, it was found that the lipid analysis dataindicated a similar composition of grapefruit exosomes andmicroparticles, i.e., similar ratios of phosphatidylethanolamine (PE) :phosphatidylcholine (PC) : phosphatidylinositol: phosphatidylserine:monogalactosyldiacylglycerol : digalactosyldiacylglycerol:LysoPC: LysoPE= 35:35:10:3:5:2:2:2).

To then produce grapefruit exosomal or microparticle liposomes thatdeliver siRNA and mammalian expression vectors, two ml of grapefruitexosomes or microparticles in PBS were transferred into a glass vial,7.5 ml of CHCl₃ added, the mixture vortexed for 1 min, 2.5 mil of MeOHadded, the mixture vortexed again, and finally 2.5 mil of water added.The mixture was then centrifuged at 500 × g for 10 min at 22° C. and thelower phase carefully collected. The extracted sample was subsequentlydried at 100° C. under N₂ gas, and the dried lipids were dissolved in100 µl CHCl₃ and transferred to a glass bottle. The lipid was dried asbefore, and siRNA, miRNAs or mammalian expression vectors in PBS wereadded as experimentally desired, with the mixtures being sonicated in awater sonication bath for 40 min to prepare the samples for transfectionof cells.

For the cell culture, the 4T-1 cell line, murine mammary adenocarcinomaof spontaneous BALB/c origin, GL-26 murine microglioma tumor cells, CT26murine colon cancer cell lines, RAW 264.7 murine macrophage, A549 humanlung carinonoma cell line, MDA-MB-231 human breast cancer cell line, and293 human kidney cell line were purchased from ATCC and maintained invitro at 37° C. in a humidified 5% CO₂ atmosphere in air in completemedium (Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum)as described previously. The fetal bovine serum used in cell cultureswas exosome depleted by differential centrifugation using a methoddescribed previously.

The DNA plasmid, pEGFP expressing green fluorescent protein withcytomegalovirus promoter was purchased from Clontech (Palo Alto, CA).The plasmid was purified using a Qiagen (Valencia, CA) kit according tothe manufacturer’s instructions. The plasmid purity was assessed bymeasuring the A₂₆₀/A₂₈₀ ratio and confirmed by agarose gelelectrophoresis. siRNAs including luciferase siRNA and scramble siRNAwere purchased from Ambion Inc.

To transfect the cells, the cells were seeded at a density of 2×10⁵cells per well onto 6-well plates (Nunclon) in 2-ml growth medium. At 24h of culture time the cells were about 80% confluent and used fortransfection. The grapefruit-derive liposome/DNA complexes or thegrapefruit-derived liposome/siRNA complexes prepared as described abovewere then added to the media drop by drop up to a volume of 100 µl.Plasmid DNA (4 µg/well) or siRNA (1 µg/well) was used.

To transfect primary spleen T cells, spleen T cells obtained fromC57BL/6j mice were isolated. Purity of the spleen T cells was routinelybetween 95 and 98% as determined by flow cytometry. Total T cells weretransfected with 4 µg of plasmid DNA at a cell density of 1 × 10⁷ cellsper 100 µl of solution using FUGENE® HD, LIPOFECTAMINE® 2000, orgrapefruit liposome methodology. After transfection, cells weretransferred immediately into prewarmed RPMI1640 culture mediumsupplemented with 2 mM glutamine, penicillin (100 U/ml), streptomycin(100 µg/ml), and 10% fetal calf serum in the presence of recombinantmouse interleukin-2 (50 u/ml) plus anti-CD3 (100 ng/ml). Forty-eight hrafter the transfection, transfection efficiencies were determined byflow cytometry analysis of GFP cells.

48 hr after cells were transfected with a reporter plasmid encoding theGFP from either grapefruit liposome or two commercial reagents (FUGENE®HD and LIPOFECTAMINE® 2000), cells were detached from the culture wellusing PBS containing 0.02% EGTA and 1 µg/ml propidium iodide. Propidiumiodide was included to identify the nonviable cells. The percentages ofdead cells were determined by flow cytometry, evaluated with theCellquest software (BD Biosciences, San Jose, CA). The percentcytotoxicity following transfection was calculated as follows: (numberof nontransfected adherent or total number of cells when grown insuspension present at the time of harvest - the number of adherent ortotal number of cells when grown in suspension in the transfected sampleat the time of harvest. The transfection efficacy was determined as the:number of EGFP positive cells/total numbers of cells.

GL26 murine microgliomas stably transfected with Lent-luc were alsotransfected with luciferase siRNA or scramble siRNA (Ambion) as anegative control. 48 hr after transfection, the cells were lysed withlysate buffer (Promega luciferase assay system kit, Madison, WI).Luciferase activity in each well was determined using a luminometer(Applied Biosystems, Foster City, CA) and the luciferase assay systemkit (Promega) according to the instruction manual. The ratio of fireflyluciferase activity of cells transfected with luciferase siRNA toscramble siRNA was calculated. An asterisk (*) indicates a p-value ofless than 0.05 in a Student’s t-test.

The ability to transfect primary immune cells opens up manypossibilities for the study of disease and the development of therapiesthat modulate the host immune response. However, there is no agentavailable to effectively transfect primary immune cells, in particular Tcells. Lentiviral vector has potential to be used as a delivery vehiclefor T cells; however the major drawbacks of using lentiviral vectorinclude insufficient transduction efficiency, putative aberrantexpression near integration sites raising safety issues and the highcost for its production. The data obtained from the experimentsdescribed herein above indicate that grapefruit-derived liposomes, whichconsist of 100% natural components of this often consumed fruit, areeffective and result in more than 80% transfection efficiency of GFPexpression in primary T cells (FIGS. 12A-12C) isolated from B6 spleenwith only 5% cytotoxicity. In comparison, only 5.3 - 0.3% transfectionefficiency was achieved when FUGENE® HD or LIPOFECTAMINE® 2000 were used(FIGS. 12A-12C) and a higher cytotoxicity resulted (10 -15%, FIG. 12D),respectively.

The transfection efficiency of other types of cells listed in FIG. 13Awas also tested. Most of the cell types have a much highersusceptibility of being transfected with grapefruit liposome than withFUGENE® HD or LIPOFECTAMINE® 2000. This higher transfection efficiencywith grapefruit liposomes was also associated with lower cytotoxicity(FIG. 13B). These results were though to not be due to poor quality ofthe other two transfection agents since all three agents have similartransfection efficiency in 293 cells.

To determine whether grapefruit liposomes were suitable for delivery ofsiRNA, the GL26 murine microglioma cell line stably expressing thefirefly luciferase gene (GL26-luc) was used. The GL26-luc cells weretransfected with either luciferase siRNA or scramble siRNA. Results fromluciferase assays showed that transfection with luciferase siRNAsignificantly repressed luciferase activity of the luciferase reporterby about 60% when compared to the control siRNA (FIG. 14 ). In contrast,there was very limited luciferase activity repression when FusionHD orLIPOFECTAMINE® 2000 was used for transfection (FIG. 14 ).

Based on the data described herein using grapefruit-derived liposomes,it was believed that the liposomes have a number of advantages overother transfection agents, including: a much higher transfectionefficiency of immune cell; a more simple, rapid and inexpensiveprotocols for making grapefruit liposomes to package nucleic acids; theability, unlike other transfection agents, to increase the transfectionefficiency by adding grapefruit liposome to the cells repeatedly withoutcausing cytotoxicity; and the presence of sera not affecting thetransfection efficiency when using grapefruit-derived liposomes in vivo.Thus, the foregoing study further demonstrated that grapefruit liposomesare effective for a broad range of cell lines and cell types, resultingin high levels of transgene expression and low cytotoxicity that canthen be used for therapeutic applications for in vivo gene delivery.

Materials and Methods for Examples 11-16

Isolation and purification of Grapefruit nanoparticles. Grapefruits withthe skin removed manually were pressed and the collected juice wasdiluted in PBS, differentially centrifuged and the nanoparticles thenpurified on a sucrose gradient. The purified nanoparticles were preparedfor EM using a conventional procedure and observed using an FEI TecnaiF20 electron microscope operated at 80 kV at a magnification of 15,000xand defocus of 100 and 500 nm. Photomicrographs were taken using an AMTcamera system.

Extraction of lipids from grapefruit-derived nanoparticles andreassembling nano-sized particles. Total lipids were extracted fromsucrose band (FIG. 15A) of processed grapefruit nanoparticles. Briefly,3.75 ml 2:1 (v/v) MeOH:CHCl₃ was added to 1 ml of grapefruitnanoparticles in PBS, and vortexed. CHCl₃ (1.25 ml) and ddH₂O (1.25 ml)were added sequentially and vortexed. The mixture was centrifuged at2,000 rpm for 10 min at 22° C. in glass tubes to separate the mixtureinto two-phases (aqueous phase and organic phase). For collection of theorganic phase, a glass pipette was inserted through the aqueous phasewith gentle positive-pressure and the bottom phase (organic phase) wasaspirated and dispensed into fresh glass tubes. The organic phasesamples were aliquoted and dried by heat under nitrogen (2 psi). Totallipids were determined using the phosphate assay as describedpreviously.

Mice. C57BL/6j mice and BALB/c mice, 6-8 weeks of age were obtained fromJackson Laboratories. All animal procedures were approved by theUniversity of Louisville Institutional Animal Care and Use Committee.

Cell culture. The mouse 4T1 breast cancer, CT26 colon cancer, and humanA549 lung epithelial cancer cell lines were purchased from ATCC. Themouse (H-2^(b)) glioblastoma cell line GL26 stably expressing theluciferase gene (GL26-Luc) was provided by Dr. Behnam Badie (BeckmanResearch Institute of the City of Hope, Los Angeles, CA), and maintainedin RPMI 1640 media supplemented with 10% heat-inactivated FBS in ahumidified CO₂ incubator at 37° C. Transient transfections of siRNA orplasmid DNAs were performed using LIPOFECTAMINE® 2000 (Invitrogen)according to protocols provided by the manufacturer.

Reagents and antibodies. Curcumin, JSI-124 (cucurbitacin I),beta-glucan, chloropromazine, indomethacin, nocodazole, cytochalasin D,bafilomycin A1, paclitaxel, and folic acid were purchased fromSigma-Aldrich (St Louis, MO) and dissolved in DMSO as stock solutions.Antibodies against total and phospho-Stat3 were purchased from CellSignaling Technology Inc. (Danvers, MA). Antibody against mouse β-actinwas purchased from Santa Cruz biotechnology (Santa Cruz, CA). Thefollowing fluorescent conjugated Abs were obtained from e-Bioscience:anti-CD4, anti-CD8 and anti-CD19. For FACS analysis of cell apoptosis,an Annexin-V fluorescein isothiocyanate/PI double-staining assay wasperformed according to the manufacturer’s protocol (BioVision, MountainView, CA). siRNA targeting the luciferase gene was purchased from LifeTechnologies (NY, USA) and the Label IT Biotin-DNA kit for biotinylationof pEYFP-C1 (Clontech) was purchased from Mirus Bio LLC (Pittsburgh,PA). Cell viability was assessed via measurement of cellular ATP levelsusing the ATPLite luminescence-based assay (Perkin Elmer, Waltham, MA).EPNVs were labeled with near-infrared lipophilic carbocyanine dye(1,1′-dioctadecyl-3,3,3′3′-tetramethyl-indotricarbocyanine-iodide, DIR,Invitrogen, Carlsbad, CA) or PKH26-GL (Sigma-Aldrich, St. Louis, MO)using a method described previously.

Lipidomic analysis. The lipid composition of EPNVs was determined usinga triple quadrupole mass spectrometer (Applied Biosystems Q-TRAP,Applied Biosystems, Foster City, CA). The data were reported as % oftotal signal for the molecular species determined after normalization ofthe signals to internal standards of the same lipid class.

Confocal image analysis of localization of EPNVs. Tumor cells (4T1,GL26, A549, CT26 or SW620) were plated on 4-chamber slides (Tissue-Tek,Sakura, USA) Lab-Tek II) and cultured at 37° C. for 24 h. Then, thecells were cultured with fresh culture media in the presence ofPKH26-labeled EPNVs (10 nmol). At variable time points after co-culturewith PKH26-labeled EPNVs, the cells were fixed with 2% paraformaldehydein PBS for 20 min at 22° C. The fixed cells were permeabilized with 0.2%Triton X-100 for 15 min, stained with 4′,6-diamidino-2-phenylindole(DAPI,) for 90 s. PKH26-loaded EPNVs in the cells were examined using aNikon A1R-A1 confocal microscope equipped with a digital image analysissystem (Pixera, San Diego, CA). For analysis of localization of EPNVs inprimary lymphocytes, freshly purified splenic T or B cells (5×10⁶) wereco-cultured with PKH26-labeled EPNVs in a 24-well tissue culture platefor 6 h at 37° C. After washing with PBS 3x, the cells were fixed,permeabilized and stained with DAPI using the identical protocol asdescribed above. Washed cells were centrifuged onto slides andPKH26-labeled EPNVs in the cells were examined using a Nikon A1R-A1confocal microscope equipped with a digital image analysis system(Pixera, San Diego, CA). To determine the effects of temperature onEPNVs uptake, A549 cells were cultured in 4-chamber slides at 37° C.,20° C. or 4° C. for 6 hours with PKH26 loaded EPNVs. After washing 3x,EPNV positive cells were observed using confocal microscopy.

Flow cytometry assay for uptake quantification of grapefruit derivedEPNVs. For uptake experiments, tumor cells were grown in 12-well plateswith Eagle’s minimal essential medium (EMEM) in the presence of 10%fetal bovine serum (FBS) for 24h. PKH26-labeled EPNVs (10 nmol/ml)freshly prepared under sterile condition were added to the culture mediaand incubated with cells for an additional 6 h. After washing with coldPBS 5x, cells were trypsinized with 0.25% Trypsin-EDTA (Invitrogen,Carlsbad, CA) and washed an additional two time. Finally, the cells wereresuspended in flow cytometry buffer and analyzed by flow cytometry (BDACCURI™ C6 Cytometer, New Jersey USA) and FlowJo Version 7.6 software(TreeStar Inc). The data presented were based on the mean fluorescencesignal for 50,000 cells collected. All assays were performed intriplicate.

To study EPNV taken up by primary lymphocytes, subsets of T and B cellswere purified from the spleens of C57BL/6j mice with CD3 and CD19 beads,respectively (Miltenyl Biotec) according to the manufacturer’s protocol.In brief, spleens were removed aseptically and splenocytes were obtainedby gently pressing the spleens between two sterile glass slides and thenwashing the lymphocytes from the slides using 10 ml of RPMI 1640 mediumcontaining 10% fetal calf serum (FCS). This was pipetted several timesand filtered through a 70-µm cell strainer (Falcon). The filtrate wasthen centrifuged at 1200 rpm for 5 min with 10% FCS-RPMI used forisolation of CD3⁺ T cells and CD19⁺ B cells according to the protocolprovided (Miltenyl Biotec). Purified CD3⁺ T cells or CD19⁺ B cells werethen resuspended and washed in RPMI 1640, cells were cultured in RPMI1640 (Invitrogen) supplemented with 10% FCS, 2 mM L-glutamine, 100 U/mlpenicillin, 100 µg/ml streptomycin, 25 mM Hepes, 50 µM2-mercaptoethanol, 20 µg gentamicin and 1 mM sodium pyruvate in thepresence of PKH26-labeled EPNVs for 6 h at 37° C./5% CO₂ incubator. Theco-cultured cells were then washed with PBS three times. The percentagesof PKH26⁺ cells were quantified by FACS analysis.

To investigate the effect of pH values on uptake efficiency, A549 cells(5X10⁵) were seeded in 6-well plates and cultured for 24 h. The culturemedium was replaced with fresh medium with different pH values (5.5,6.5, 7.4 and 9.0) and culturing continued with PKH26-labeled EPNVs (10nmol/ml) for 6 h. The cultured cells were then washed with PBS 3x. Thepercentages of PKH26⁺ cells were quantified by FACS analysis.

To study the effect of endocytosis inhibitors on EPNV uptake, cells werecultured at 37° C. in the presence of an endocytosis inhibitor for 1 hprior to the addition of PKH26-labeled EPNVs, for an additional 6 hculture period. The cultured cells were then washed with PBS 3x. Thepercentages of PKH26⁺ cells were quantified by FACS analysis (BD ACCURI™Flow Cytometer) and FlowJo Version 7.6 software (TreeStar Inc.).

To analyze the effects of temperature on EPNVs uptake, A549 cells werecultured in 6-well tissue culture plates with PKH26 loaded EPNVs at 37°C., 20° C. or 4° C. for 6 hours. After washing 3x, PKH26+ cells wasanalyzed using FACS.

To determine whether the uptake of EPNV by A549 cells was energydependent, confluent A549 cells were exposed to PKH26 labeled EPNVs (10nmol/ml) for 3 and 6 hours at 37° C. in the presence or absence of ametabolic inhibitor--50 mM sodium azide. After washing 3x with PBS(pH7.4), PKH26⁺ cells were determined by FACS analysis as previouslydescribed³. Cells exposed to the vehicle (PBS; pH 7.4), served as acontrol for intrinsic fluorescence, both in the presence and the absenceof 50 mM sodium azide. The data were analyzed by FACS (BD ACCURI™ FlowCytometer) and FlowJo Version 7.6 software (TreeStar Inc).

To identify the cells that EPNVs targeted in vivo, mice wereintravenously injected with PKH26 labeled EPNV (200 nmol/mouse). 72 hafter injection, total spleen and liver cells resuspended in FACSanalysis buffer were stained with anti-CD4, CD8, CD19, DX5, and F4/80antibodies for further quantitatively analysis of PKH26⁺ cells.

Fluorescent imaging in vitro and vivo. In vitro imaging assays for A549cells were conducted with EPNVs. A total of 1X10⁵ cells were added toeach well of 12-well plates. DIR dye-loaded EPNVs were diluted in PBSand added to 3 wells per concentration. After incubation at 37° C. in 5%CO₂ at variable times, cells were washed with ice cold PBS 5x. Lightemission from the wells of the plates was measured with a Kodak ImageStation (4000MM Pro system, Carestream, Woodbridge, CT) and quantifiedusing the vendor software. Regions of interest (ROI) were drawn manuallyaround the area of each individual well of plate, and the intensity oflight emitted from each ROI was measured. Data were normalized to lightemission of an equal number of untreated cells otherwise incubated underthe same conditions as the treated cells.

To evaluate the stability of circulating EPNVs in mice, 200 nmol of DIRdye-encapsulated EPNVs were injected via tail vein. At various timepoints (1 h, 2 h, 6 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h and 172 h),blood was withdrawn into an anti-coagulant tube. The DIR signals fromequal volume blood samples were then measured with a Kodak Image Station(4000 MM Pro system, Carestream, Woodbridge, CT) and quantified usingthe vendor software.

To determine the biodistribution of EPNVs in mice administered bydifferent routes, 100 nmol of EPNVs were administered (subcutaneously,intraperitoneally, intravenously, and intramuscularly, and by intranasaldrop) using methods as described previously. 72 h after administrationmice were sacrificed and each organ was collected and light emissionfrom the samples was measured with a Kodak Image Station (4000MM Prosystem, Carestream, Woodbridge, CT) and quantified using the vendorsoftware.

To evaluate the stability of injected EPNVs, mice were intravenouslyinjected with 100 nmol of DIR dye labeled EPNVs and sacrificed atdifferent time points (1 d, 3 d, 5 d, 7 d, 9 d, 12 d, 15 d, 20 d). Lightemission from each organ of mice was then measured using an Odysseylight imager and quantified using the vendor software.

Assessment of liver damage and HE stained tissues. ALT and aspartateaminotransferase (AST) in the sera of the mice were quantitativelyanalyzed using the Infinity Enzymatic Assay Kit (Thermo Scientific). Forhistopathology analysis, H&E staining was performed on paraffin-embeddedliver, lung, spleen, and kidney sections using a method as described.

Western blot. Western blots were done by first lysing cells and then theproteins of the lysed cells were separated on 10% polyacrylamide gelsusing SDS-PAGE. Separated proteins were transferred to nitrocellulosemembranes. The western blot was carried out with the anti-Stat3 andanti-phospho-Stat3 antibodies (Cell Signaling) or anti-β-actin antibodyas a control (Santa Cruz Biotechnology, Santa Cruz, CA).

Cytokines assay. To determine the effect of EPNVs or EPNV encapsulatedcurcumin on the induction of cytokines, and splenocytes (5X10⁶),C57BL/6j mice were pre-stimulated with LPS (10 µg/ml) for 3 h, thenEPNVs (200 nmol), free curcumin (50 µg) or EPNVs encapsulated curcumin(50 µg curcumin in 200 nmol EPNVs) was added and cultured for another 6h. TNF-α and IL-6 in culture medium were measured using ELISA kits(eBioscience).

For analysis of immune stimulation of beta-glucan loaded EPNVs,splenocytes (5X10⁶) from C57BL/6j mice were cultured in 6-well plates inthe presence of free beta-glucan (20 µg) or EPNV-beta-glucan (20 µgbeta-glucan in 200 nmol EPNVs) for 6 h. then TNF-α and IFN-y insupernatants were measured using ELISA kits (eBioscience).

Analysis of storage stability of EPNVs. To determine the storagestability of agents carried by EPNVs, free PKH26-GL labeled andcurcumin-loaded EPNVs were prepared in PBS (pH7.4) and stored at 4° C.The stability and bioactivity of curcumin was determined by theanti-inflammatory function of curcumin over a 2 month period.

In vitro and in vivo cytotoxicity analyses. For evaluation of in vivotoxicity of injected EPNVs, mice were injected intravenously with 100nmol of EPNVs once per day for 1 or 5 days. 24 h after the lastinjection, sera were collected for quantification of cytokines (TNF-α,IL-6, IL-10, IFN-γ and TGF-β) using a standard ELISA kit (e-BioScience)and liver ALT and AST were analyzed using AST/ALT Liquid Stable Reagent(Thermo Scientific).

For evaluation of in vitro toxicity of EPNVs, ATPLite assay was used foranalysis of A549 cell viability and the Annexin V-FITC/ Propidium iodideapoptosis assay was used for quantifying percentages of death of A549cells treated with EPNVs. Briefly, 1×10⁵ A549 cells were cultured in a12-well plate at 37° C. under 5% CO₂ atmosphere for 24 h. EPNVs at thedifferent concentrations (2, 4, 8, 20 and 40 µM) were added to the 24-hcultured media and the cells were cultured for additional 48 h. Thepercentage of viable cells was then determined using the ATPlite assayfollowing the protocol provided by the manufacturer (Perkin Elmer). Allexperiments were conducted in triplicate.

The Annexin V-FITC/ Propidium iodide apoptosis assay was used toquantify cell death in vitro. A549 cells were placed in a six-wellculture plate and cultured in the presence of different concentrations(4, 8, 20, 40 and 80 µM) of EPNVs for 24 h. Cultured A549 cells werewashed with ice cold PBS three times. Cells were harvested bytrypsinization and stained using an Annexin V/FITC Apoptosis Detectionkit (Roche, Cambridge, MA) according to the manufacturer’s protocol. Thestained cells were immediately analyzed by flow cytometry (FACScan;Becton Dickinson, Franklin Lake, NJ).

Imaging EPNVs across the placental barrier. To determine whether theEPNVs passed through the placental barrier of pregnant mice and diffusedinto the fetus, pregnant C57BL/6 mice were injected intravenously withDIR dye labeled EPNVs daily for 1 or 5 days (50 nmol of finalphospholipid at each injection/mouse, n=5). 72 h after the lastinjection, the fetus and placenta were removed from anesthetizedpregnant mice and imaged using the Odyssey image system or a Kodak ImageStation.

Purification of drug/chemicals/DIR dye or siRNA encapsulated EPNVs. Achemotherapy drug, JSI124, paclitaxel or agents including folic acid,zymosan A or luciferase gene siRNA were mixed with total lipids fromgrapefruit dissolved in chloroform and dried under nitrogen to obtain athin and dried lipids-complex film. The film was reconstituted in PBSbuffer, followed by sonication in water-bath sonicator for 30 min,allowing the lipids to self-assemble into drugs/chemicals/siRNA-loadednanoparticles. Then drug/chemicals/siRNA loaded EPNVs were then purifiedvia sucrose gradient. The purified band was collected and washed at100,000xg for 2 h before use.

For preparation and purification of EPNVs loaded with biotin labeledanti-CD4, CD8 antibodies or eYFP vectors, biotin labeled anti-CD4, CD8antibodies (2.5 µg, BD Pharmingen, USA) were incubated with EPNVs (200nmol) at 4° C. overnight. The EPNVs-biotin-anti-CD4 orEPNVs-biotin-anti-CD8 complex was washed with PBS at 36,000 rpm for 2 hand the pellet resuspended in PBS for the transfection of T cells. Toprepare eYFP vector loaded EPNVs, biotin labeled eYFP vectors (5 □g)were incubated with EPNVs (200 nmol) in OPTI-MEM at 37° C. for 2 h andsubsequently used for transfection.

Brain tumor-bearing mice model. GL26-luc brain tumor-bearing mice wereprepared as reported previously. Tumor-bearing mice were treatedintranasally for 10 consecutive days with EPNVs JSI124 (12.5 pmol) orJSI124 loaded EPNVs. GL26 tumor growth was monitored by quantifying theactivity of luciferase activity. Images were collected using ahigh-sensitivity CCD camera with wavelengths ranging from 300 to 600 nmwith an exposure time for imaging of 2 minutes. Regions of interest wereanalyzed for luciferase signals using a Kodak Image Station and reportedin units of mean intensity.

In vivo imaging of EPNV mediated targeting in tumor models. Xenografttumor growth models were used to demonstrate EPNV mediated targeteddelivery of chemotherapy drug to tumors versus standard chemotherapywith paclitaxel. In the first set experiments, six-week-old femaleBALB/c-SCID mice (Jackson Lab) were injected subcutaneously with thehuman colon cancer SW620 cell line (5.0×10⁶ cells/mouse in 50 µl ofPBS). In the second set of experiments, six-week-old female BALB/c mice(Jackson Lab) were injected subcutaneously with murine colon cancer CT26cell line (1.0 ×10⁶ cells/mouse in 50 µl of PBS). In the third set ofexperiments, six-week-old female BALB/c mice (Jackson Lab) were injectedat a mammary fat pad with the murine breast tumor 4T1cell line (1.0 ×10⁶cells/mouse in 50 µl of PBS). When tumors reached approximately 60 mm³in volume, the mice were randomly assigned to different treatment groupsand injected intravenously with free EPNVs, paclitaxel (PTX, 20 mg/kg),EPNVs (200 nmol) loaded with folic acid (5 µg, EPNVs-FA), EPNVs (200nmol) loaded paclitaxel (20 mg/kg, EPNVs-PTX) and EPNVs (200 nmol)loaded folic acid plus PTX (EPNVs-FA-PTX). Mice were treated every 3days for 30 days with the last injection of being DiR dye labeled EPNVs.Growth of the tumors was measured. Biodistribution of EPNVs wasmonitored using a Kodak Image System after the final intravenousinjection. Mice were sacrificed, tumors and other organs were removedand biodistribution of DiR labeled EPNVs was analyzed with a Kodak ImageSystem.

In vivo imaging of EPNV mediated targeted delivery of siRNA model.CT26-luc tumor bearing mice were prepared as described above andinjected intravenously with free EPNVs, folic acid loaded EPNVs, EPNVsencapsulated with luciferase gene siRNA or both folic acid andluciferase gene siRNA every 3 days for a total of 5 injections. Beforestarting the imaging, mice were intraperitoneally administratedD-luciferine (150 mg/kg; Xenogen, Alameda, CA) dissolved in PBS and thenanesthetized for determining the intensity of the mouse luciferasesignals using a Kodak Image Station.

Example 11 - Characterization of Nano-Sized Particles Assembled FromEdible Plant-Derived Nanoparticles Lipids

Using the foregoing techniques, edible plant nanoparticles were isolatedfrom the juice of grapefruits. The particles were identifiable asnanoparticles based on electron microscopic examination (FIG. 15A,right) of a sucrose gradient purified band (FIG. 15A, left).Nanoparticles purified from grape and tomatoes were also identified byelectron microscopy. Juices from edible plants were enriched innanoparticles (0.8 ± 0.07 g/pound of grape, 1.0 ± 0.02 g/pound ofgrapefruit, and 0.2 ± 0.01 g/pound of tomatoes), indicating that certainedible plants could serve as a resource for large scale production offruit derived nanoparticles.

To determine whether lipids from grapefruit nanoparticles could bereassembled into nano-sized particles for use as a delivery vector,grapefruit nanoparticle derived lipids were used and are referred tohereafter as an edible plant-derived nano vector or EPNV. Based onelectron microscopic examination (FIG. 15B, right) of a sucrose gradientpurified band (FIG. 15B, left) and the lipid profile (FIG. 15C, FIG. 20), the reassembled nano-sized particles were similar to the grapefruitnanoparticles. Nanoparticles assembled from the lipids of two othersucrose gradient bands (band 2 and 4, FIG. 15A, left) were alsoprepared, but a single uniform band could not be obtained (FIG. 20 ).Although the nanoparticles generated initially were heterogenous insize, passing the nano particles through a homogenizer resulted in moreuniform sized nanoparticles (FIG. 15D). Electron microscopy resultsshowed that most of the reassembled nanoparticles had a multi-layerflower-like structure (FIG. 15E). Collectively, these results indicatedthat lipids derived from grapefruit nanoparticles could be reassembledinto nano-sized particles and in large quantities.

Example 12 - Uptake and Toxicity of EPNVs

To evaluate the potential use of EPNVs as a vector to delivertherapeutic agents, the tropism and toxicity was evaluated. Theefficient uptake of EPNVs by different cell types was first evaluated.The following cells were used: glioma cell line GL26 (murine brainmacrophages), lung cancer cell line A549 (human lung epithelial cell),SW620 (human colon cancer cell line), CT26 (murine colon cancer cellline), 4T1 (murine breast cancer cell line), and primary mouse T cellsand B cells (splenic lymphocytes) isolated from BALB/c mice. Each of thecell types was co-cultured with PKH26 labeled EPNVs (PKH26-EPNVs) andthe presence of EPNVs in cells was examined using confocal microscopy(FIG. 16A) or by FACS (FIG. 16B), and determined by quantitativeanalysis of PKH26-EPNVs⁺ cells. The results indicated that the majorityof GL26, A549, SW620, CT26, and 4T1 cells took up the EPNVs. More than20% of the B cells and 14% of the T cells took up the EPNVs within 12hours after the co-culture, which was remarkable since B and T cells areconsidered the most difficult to transfect using anycommercially-available transfection agents. When comparing the resultswith cells incubated with free PKH26 dye, distinct patterns of PKH26⁺staining were observed in cells incubated with PKH26 labeled EPNVs whichwas not the case in cells incubated with free dye (FIG. 21 ). Thisindicated that the PKH26⁺ signals were derived from the EPNVs⁺ cells,not with free PKH26 dye contamination. Using A549 as an example, it wasfurther demonstrated that the efficiency of uptake of EPNVs was atemperature-dependent process. Uptake rates were very slow at 4° C. andincreased as the temperature was raised (FIG. 16C). The results fromimaging (FIG. 16C, top panel) or from FACS analysis (FIG. 16C, bottompanel) of A549 co-cultured with PKH26 labeled EPNVs indicated that morethan 87% of A549 cells took up the EPNVs at 37° C., but not at 20° C. orat 4° C. Uptake of EPNVs at 37° C. in the presence of the metabolicinhibitor sodium azide (50 mM) was significantly reduced after 3 and 6hour incubations (FIG. 22 ), suggesting that metabolic energy isrequired for this process. Under physiological temperature (37° C.)conditions, an initial rapid uptake of DiR dye labeled EPNVs (20 nmol)was observed within the first 2 h (the first time point) and followed bya linear uptake that reached a peak between 20 to 24 h (FIG. 16D). Theuptake of DiR dye labeled EPNV by A549 cells was also found to be EPNVconcentration dependent. Treatment with the highest concentration (40nmol/ml) of EPNVs resulted in no reduction of EPNV uptake (FIG. 16E),indicating that epithelial A549 cells have a high capacity for taking upEPNV. To further examine the mechanism of EPNV internalization, A549cells were treated with endocytosis inhibitors. Uptake of PKH26-EPNV(FIG. 16F) was markedly inhibited by the macrolide antibioticbafilomycin A1, which prevents maturation of autophagic vacuoles. Inaddition, uptake of PKH26-EPNVs was greatly diminished by treatment ofA549 cells with cytochalasin D, an inhibitor of microfilament formationrequired for phagocytosis, nocodazole, an inhibitor of thepolymerization of microtubules, and the clathrin-mediated endocytosisinhibitor chlorpromazine. Amiloride, an inhibitor of macropinocytosis,and the caveolae-mediated endocytosis inhibitor indomethacin did notaffect uptake of PKH26-EPNV. Increasing the pH from 6.5 to 9.0 had noapparent effect on the uptake of EPNV (FIG. 23 ). Whether EPNVs weretoxic to A549 cells was next determined. The results of the ATPliteassay, which quantitatively measures cell proliferation, and thePI/Annexin V assay, which quantifies cell death, revealed that EPNVtreatment at concentrations up to 40 nmol/ml has no effect on A549 cellproliferation (FIG. 24A) or death rates (FIG. 24B) when compared withPBS treated cells. Collectively, these findings indicated that underphysiological temperature conditions, EPNVs were taken up by both celllines tested, as well as primary lymphocytes. Moreover, EPNVs werestable at 4° C. for more than 1 month and did not lose their ability tocarry curcumin and aid in maintaining the biological activity ofcurcumin as determined by its persistent anti-inflammatory activity(FIG. 17A). Based on these results, it was believed that EPNVs had thecapacity to deliver therapeutic products in vitro.

Example 13 - Tissue Tropism of EPNVs

To determine the tissue tropism of EPNVs, in vivo biodistribution ofDiR-labeled EPNV was evaluated in mice using a Kodak Image Station4000MM Pro system or the Odyssey imaging system. For these studies, theeffect of different routes of injection on the distribution ofDiR-labeled EPNV was first evaluated. 72 h after a tail-vein orintraperitoneal injection, DiR fluorescent signals were predominantlydetected in liver, lung, kidney, and splenic tissues (FIG. 17B);whereas, intramuscularly injections of the DiR-labeled EPNVs werepredominantly localized in muscle. After intranasal administration (FIG.17C) of DiR-labeled EPNV the majority of the nano-vector was located inthe lung and brain. The presence and intensity of the imaging signalfurther indicated that DiR-labeled EPNVs remain stable in the brain;whereas, no signal was detected in lung tissue 72 h after intranasaladministration. FACS analysis was done on cells from mice receiving anintravenous injection of DiR-labeled EPNVs. FACS analysis indicated thatEPNVs injected intravenously were taken up by splenic DX5⁺ NK cells(10.9%) and F4/80⁺ cells (12.7%), and liver F4/80⁺ cells (4.65%), DX5⁺NK (1.75%), and CD19 B cells (1.63%) (FIG. 17D) 72 h after injectionUpon analysis of the stability of intravenously injected DiR-labeledEPNVs, it was found that the fluorescent signals remained strong withouta significant decrease in liver, spleen, and lung, while the signalswere decreased significantly in the kidney at day 1 and in the brain atday 5 (FIG. 17E). In vivo imaging to continuously track the stability ofinjected DiR-labeled EPNVs further revealed that fluorescent signalsremained strong in the liver and spleen at day 20 (FIG. 17E).Surprisingly, circulating DiR-labeled EPNVs were still detectable 7 daysafter a tail-vein injection (FIG. 17F). More importantly, unlikeartificial nanoparticles that cross the placenta barrier in pregnantmice and cause pregnancy complications, in vivo imaging analysis showedthat mice tail-vein injected with DiR labeled EPNVs had no EPNVs passingthrough the placenta (FIG. 17G), indicating that EPNV treatment can besafe for the fetus. Collectively, these data indicate that the stabilityof EPNVs is dependent on the microenvironment of tissues the EPNVs hometo, and that the extended duration of circulating EPNVs can provide anopportunity for EPNVs to be available for a longer time in circulationand provide more time for the vector to eventually make it to itstarget.

Example 14 - Assessment of in Vivo Cytotoxic Effects of the EPNVs

To further explore the potential in vivo cytotoxic effects of the EPNVs,proinflamamtory cytokines and indicators of liver injury werequantitatively determined. Serum levels of alanine aminotransferase(ALT) and aspartate aminotransferase (AST) of mice pre-treated withEPNVs were measured for liver injury. ALT, AST (FIG. 25A) andproinflammatory cytokines (FIG. 25B) were not induced due to EPNVtreatment. Histological analysis of tissues from treated animals andcontrol animals (FIG. 25C) revealed no pathological changes in the lung,kidney, liver, or spleen. Hepatocytes in the liver samples appearednormal, and there were no signs of inflammatory response. No pulmonaryfibrosis was detected in the lung samples. Necrosis was not found in anyof the histological samples analyzed.

Example 15 - EPNVs as Candidates for Use as a Therapeutic DeliveryVector

The molecular or drug therapy fields are limited by the lack of vehiclesthat permit high efficiency transfection of targeted cells without aresulting cytotoxicity or host immune response. The results presented inFIGS. 16A-F and FIGS. 25A-C demonstrated that EPNVs were taken up in ahighly efficient manner by a number of different types of cells withoutcausing cytotoxicity or an inflammatory cytokine induction. Next, it wasdetermined whether EPNVs can deliver a broad range of the therapeuticagents such as chemotherapeutic drugs, siRNA, DNA expression vector, andproteins to targeted cells. Previous data suggested that nano sizedparticles released from mammalian cells favor binding to hydrophobicagents, such as curcumin and anti-stat3 JS124, resulting in increasedstability, solubility, and bioavailability of the drugs. Resultspresented in this study also show that EPNVs bind to hydrophobic agentsincluding curcumin, folic acid, and beta-glucan without altering thebiological activities of the agents (FIG. 26 ). To further determinewhether EPNVs can also carry an agent that can serve as a bridge todeliver therapeutic agents, biotin was chose as a candidate since biotinis a small (244.31delta), hydrophobic molecule. Biotinylized eYFP DNAexpression vector (FIG. 27 ) carried by EPNVs expressed the YFP proteinin A549 cells as efficiently when transfected with LIPOFECTAMINE® 2000(FIG. 18A). Furthermore, EPNVs carrying biotinylized proteins likeanti-CD4 or anti-CD8 antibodies significantly enhance the transfectionefficiency of CD4⁺ or CD8⁺ T cells (FIG. 18B). Collectively, theseresults indicated that EPNVs are capable of delivering both biotinylizedDNA, as well as proteins to targeted cells.

To determine whether EPNVs would encapsulate and deliver functionalsiRNAs, a well-characterized siRNA that is directed against a luciferasereporter gene stably expressed in GL26-Luc was used. Transfection wasconducted with 15 pmol luciferase siRNA delivered by EPNVs or by astandard LIPOFECTAMINE® 2000 transfection agent. It was found thatluciferase siRNA carried by EPNVs effectively inhibited the expressionof the luciferase gene when compared with the LIPOFECTAMINE® 2000untreated cells (FIG. 18C). In summary, the results showed that EPNVsare an effective delivery vector for all the agents tested.

Example 16 - In Vivo Delivery of Therapeutic Agents With EPNVs andCo-Delivery of FA and Therapeutic Agents for Targeting to Tumor Tissuesand Inhibition of Tumor Growth

It is appreciated that EPNVs are capable of carrying the anti-Stat3inhibitor, JS124, and that mice given an intranasal dose of nano-sizedmammalian cell-derived exosomes carrying JS124 had significantinhibition of GL26 tumor growth. In that regard, it was hypothesizedthat EPNVs can also deliver JS124 to the brain via a non-invasive routeand subsequently inhibit implanted GL26 tumor growth. In initialexperiments, inhibition of Stat3 activity by EPNV-JS124 or JS124 wasevaluated in 24 h cell cultures. Western blot assays revealed thatJS124-loaded EPNV significantly inhibited the activation of Stat3 incomparison with GL26 tumors treated with EPNVs only, JSI-124 only, orPBS as controls (FIG. 28 ). Based on the western blot results from cellcultures, groups of GL26L tumor-bearing mouse (n=5) were administratedintranasally EPNV encapsulated Stat3 inhibitor JSI-124 (12.5 pmol/10µl), EPNV only, JSI-124 only, or PBS. Bioluminescent imaging of the micetreated as described above was used to quantify Luc expression inrelation to the GL26 tumor growth. FIG. 19A compares brain-associatedphotons obtained from the above groups on days 5, 10, 15 and 20. Arepresentative image (left panel) or imaging data (right panel, top)showed the weakest luciferase activity relative to growth of tumors fromthe mice treated with EPNV encapsulated Stat3 inhibitor JSI-124 comparedwith other groups. These results were further confirmed by the survivalrates of mice. Survival of the PBS-, EPNVs- or JSI124-control animalsranged from 20 to 30 days. In contrast, EPNV-JSI124 treatmentsignificantly prolonged the survival of mice to an average of 42.5 ± 2.3days (P < 0.05) (FIG. 19A, right panel, bottom).

In cancer therapy accurate targeting of tumor tissue is required forsuccessful therapy. Therefore, whether EPNVs can be modified to achievetumor targeting was tested. High-affinity folate receptors (FRs) areexpressed at elevated levels on many human tumors and in almostnegligible amounts on non-tumor cells. As such, three tumor xenograftmodels (FIG. 19B) including the mouse CT26 colon cancer model, the humanSW620 colon cancer SCID mouse model and the 4T1 breast tumor model wereused to test whether EPNV binding folic acid (FA) would significantlyenhance EPNVs targeting to tumor in a physiologic milieu. EPNVs werelabeled with DiR dye for molecular imaging. 72 h after intravenousinjection of DiR dye labeled EPNVs, no DiR-labeled EPNV signals weredetected in tumor tissues with most signal being detected in the liver(FIG. 19B, the second columns from left). In contrast, intravenouslyinjected DiR-labeled EPNV-FA exhibited a much higher distribution totumor tissues (FIG. 19B, the third column from left). Co-delivery of FAwith a chemotherapeutic drug (PTX) by EPNVs has at least equalefficiency as EPNV-FA in targeting to tumor tissue (FIG. 19B),suggesting that co-delivery of FA with chemotherapeutic agents has noeffect on FA mediated targeting. Quantification of photons showed thatDiR-labeled EPNV-FA distribution to tumor tissues was more than1300-fold (CT26 model), 1600-fold (SW620), and 1400-fold (4T1 breasttumor model) greater than that of DiR-labeled EPNVs (FIG. 19B, rightpanels). Next, it was determined if co-delivery of FA with a therapeuticdrug (PTX) by EPNVs would have better therapeutic effect than the drugalone. As expected, the EPNV-FA-PTX treatment caused a substantialdecrease in tumor growth in all three tumor models. The tumor growth wassignificantly lower after treatment with EPNV-FA-PTX, an effect that wasevident from day 20 (all three tumor models) (FIG. 19C). On day 30, thetumor volume in the PTX loaded EPNV-FA group was 261.7± 28.2 mm³,significantly smaller that than in other groups (FIG. 29 ). Consistentwith the inhibition of tumor growth, FA carried by EPNVs significantlyenhanced the signal of DiR labeled EPNV-FA or EPNV-FA-PTX in tumortissues in all three tumor models (FIG. 19D), indicating that the effectis attributable to the FA. In vivo biodistribution results with EPNVs,EPNV-FA, PTX and EPNV-FA-PTX from sacrificed tumor bearing miceillustrated that free EPNVs and PTX mainly targeted liver and spleen,but FA or FA-PTX loaded EPNVs primarily targeted tumors (FIG. 20 ). Itwas further demonstrated that not only did EPNV-FA enhance thetherapeutic effect of a chemotherapeutic drug by inhibiting implantedtumor growth, but it also dramatically enhanced the efficiency ofdelivery of siRNA to tumor. As shown in FIG. 19E, intravenous injectionof EPNV-FA-siRNA-Luc led to more than a 5-fold reduction in luciferaseactivity in CT26 tumor cells compared with EPNV-siRNA-Luc under the sameconditions.

Discussion of Examples 11-16

A number of strategies, including nanotechnology, and viral andnon-viral delivery systems, have been used to experimentally determinethe most suitable vector for treatment of diseases. Each of theseapproaches has advantages. However, potential toxicity, tissue specifictargeting, hazardous effect on the environment, and large scaleeconomical production are challenging issues confronting thistechnology. The present approach of using edible plant derivednanoparticles to make a nano-vector has the advantage of havingno-detectable toxicity, the potential of being manipulated/modified forredirected targeting, the capacity to deliver varied, multiple agentsand can be produced economically.

A preparation process is required for practical large-scale generationof nanoparticles that can be loaded with multiple drugs. The present useof differential centrifugation followed by sonication allowed for largescale production of an edible fruit derived nano vector. The presentprocess is another major advantage over the multiple steps andsophisticated techniques required for in vitro synthesis of artificialor mammalian based nanoparticles. The present results show that thenano-vector produced as described can serve as the basis for developingmore customized therapeutic delivery vehicles based on the disease. Theincorporation of biotinylated therapeutic agents into a nano-vectorconsiderably broadens the range of therapeutic agents and targetingmoieties that could be delivered. A variety of substances includingprotein, peptides, nuclear acids, and chemotherapeutic agents can bebiotinylated. The vector technology presented in this study as appliedto cancer therapy can also be used for treatment of many other types ofdiseases by co-delivering therapeutic drugs with tissue specifictargeting agents.

The foregoing studies also demonstrated that chemotherapeutic drugs aswell as siRNAs can be encapsulated into the nano vector and that theirbiological effects in vivo are not then altered. This is an aspect forimproving the delivery of siRNAs/miRNAs and chemotherapeutic drugs,especially, hydrophobic drugs. Stand-alone chemotherapy drugs sufferfrom numerous problems including rapid in vivo metabolism and/orexcretion, an inability to access and penetrate cancer cells, andnonspecific uptake by healthy cells and tissue. Often a large percentageof a cytotoxic drug administered to a patient does not reach the tumorbut is distributed throughout the body, causing the numerous toxiceffects associated with chemotherapy reducing its therapeuticusefulness. In contrast, our nano-vector is derived from edible planttissue and is composed of biocompatible and biodegradable materials,encapsulates a wide range of drugs and drug classes, has the ability toattach in a targeting fashion to specific cell types or groups, protectsthe therapeutic agent from degradation and delivers the therapeuticagent directly to the site of disease.

The size of a nanoparticle is a factor that can prevent renal clearance(typically less than 20 nm), prevent uptake by the liver and spleen(particles greater than 150 nm), and enhance accumulation in the tumor(particles between 50-150 nm). One advantage of the nano-vectordescribed herein is that the size can be further manipulated by changingthe frequency at which the nano-vector passes through a high pressurehomogenizer. This allows the vector size to be tailored for specifictherapeutic treatments. An additional advantage of thepresently-described nano-vector is its retention in the circulation forextended periods. The foregoing data showed that the nano-vector wasdetected on day 7 after intravenous injection. The longer the nanovector is in circulation, the more opportunity for the ERP effect andsubsequent penetration into tumor tissues. The EPR effect in combinationwith active targeting by the nano vector would enhance the therapeuticeffect.

In summary, the foregoing studies show that specially designednano-vectors derived from edible grapefruit nanoparticle lipids couldshift the current paradigm of drug delivered by artificially synthesizednanoparticles to using nano vectors derived from edible plants. It isconceivable that nano-vectors derived from edible plants could be amongthe safest therapeutic vectors because they do not cause cytotoxicreactions. In addition, mice treated via intranasal delivery with thenano-vector carrying a therapeutic drug have a significantly delayedtumor growth in the brain, and treated intravenously, it was found thatthe nano vector does not cross the placenta to the fetus. Collectively,the data generated in this study indicated that the presently-describededible plant derived nano-vector would be safe for clinical use. Theforegoing studies demonstrate the successful inhibition of tumor growthin four independent murine cancer models using the nano vector, andindicated that it can be used as a delivery vehicle for treatment ofvarious types of cancer. Furthermore, the foregoing studies showed thata large quantity of nanoparticles can be isolated from a number ofedible plants and that a large quantity of non-toxic nano-vector can begenerated from different edible plant sources at affordable prices.

Throughout this document, various references are mentioned. All suchreferences are incorporated herein by reference, including thereferences set forth in the following list:

REFERENCES

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It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thesubject matter disclosed herein. Furthermore, the foregoing descriptionis for the purpose of illustration only, and not for the purpose oflimitation.

What is claimed is:
 1. A composition comprising a therapeutic agentencapsulated by a microvesicle, wherein the microvesicle compriseslipids extracted from an edible plant.
 2. The composition of claim 1,wherein the lipids are selected from phosphatidylcholines (PC),phosphatidylethanolamines (PE), and phosphatidic acid (PA).
 3. Thecomposition of claim 1, wherein the therapeutic agent is selected froman siRNA, a DNA expression vector, and a protein.
 4. The composition ofclaim 1, wherein the therapeutic agent is a nucleic acid.
 5. Thecomposition of claim 1, wherein the composition is formulated for oraladministration or intranasal administration.
 6. The method of claim 1,wherein the therapeutic agent is a protein.
 7. The composition of claim1, wherein the edible plant is a fruit.
 8. A method for treating adisease in a subject comprising administering to the subject aneffective amount of a composition comprising a therapeutic agentencapsulated by a microvesicle, wherein the microvesicle compriseslipids extracted from an edible plant, and further wherein themicrovesicle comprises at least 20% of phosphatidylcholines (PC) byweight.
 9. The method of claim 8, wherein the lipids are selected fromphosphatidylcholines (PC), phosphatidylethanolamines (PE), andphosphatidic acid (PA).
 10. The method of claim 8, wherein thetherapeutic agent is selected from an siRNA, a DNA expression vector,and a protein.
 11. The method of claim 8, wherein the therapeutic agentis a nucleic acid.
 12. The method of claim 8, wherein the composition isformulated for oral administration or intranasal administration.
 13. Themethod of claim 8, wherein the therapeutic agent is a protein.
 14. Themethod of claim 8, wherein the edible plant is a fruit.